Preliminary Study On Heat-resistant Mechanism Of HarpinX Protein From Xanthomonas

Harpin is a disease-resistant activator protein encoded by hrp and also a heat-resistant protein.It is secreted by type ? secretion system(T3SS)of gram-negative plant pathogenic bacteria.It can induce hypersensitive response(HR)in non-host plants and is also a pathogenic factor of pathogenic bacteria.At present,the mechanism of Harpin has been deeply studued at home and abroad.but there is no special report on its heat-resistant mechanism.In order to study the heat-resistant mechanism of HarpinX.HpaXm,Hpa1Xoo,HpaXam and HpaXcm were used as materials in this study.Using bioinformatics technology,the structural characteristics of proteins were analyzed in depth;the effects of high temperature on protein function were explored from both the surface and microscopic aspects through tobacco leaf phenotype test and quantitative real-time PCR(qRT-PCR);the effect of high temperature on protein structure was investigated by measuring the absorbance and circular dichroism(CD)spectra of protein;the coiled-coil(CC)structure was modified by site-directed mutagenesis to verify the relationship between the CC structure and the thermal stability of protein.The main results are as follows:1.The thermalstability of 4 HarpinX proteins was analyzed and compared by tobacco leaf phenotypic test.It was found that the effect of high temperature on the HR activity of Hpa1Xoo and HpaXcm was not significant,indicating that the thermal stability of Hpa1Xoo and HpaXcm was better.High temperature results in a significant decrease in the HR activity of HpaXm and HpaXam,indicating a poor thermal stability.2.By detecting the absorbance of protein,it was found that the absorbance of HarpinX increased with the increase of treatment temperature.It was speculated that high temperature led to the gradual exposure of phenylalanine embedded in the structure and the gradual unfolding of the protein structure.The contents of secondary structure were detected by CD spectroscopy.It was found that the contents of a-helix in HarpinX increased to nearly 100%after high temperature treatments.It is further confirmed that high temperature changes the HarpinX structure and promotes the transformation of other secondary structures to a-helix.3.The phenotype test showed that the HR activity of HarpinX did not change significantly with high temperatures.The relative expressions of NtEXP6,Hin1,HSR203J,NPR1 and PR-1a were detected by qRT-PCR.It was found that high temperatures caused different changes in gene expressions.It is speculated that the relationship between the thermal stability of HarpinX and the spatial structure of protein is weak,while the ability of protein to induce gene expression is closely related to the spatial structure of protein.4.Bioinformatics was used to analyze the protein.It was found that CC structure could be formed in the the a-helix region at the N-terminal of HarpinX,but the possibilities were different.The CC structures were modified by site-directed mutagenesis and mutants HpaXm?L39A,HpaXmAD40A,Hpa1Xoo?L40A,Hpa1Xoo?D41 A,HpaXam?L44A,HpaXam?M54L,HpaXcm?L40A and HpaXcm?D41 A were successfully obtained.Western blot showed that Leu44 of HpaXam was essential for soluble protein expression.Combined with the prediction of CC,it was found that the thermalstability of the corresponding mutants would be weakened if the formation of CC was destroyed;on the contrary,if the formation of CC was strengthened,the thermalstability of the corresponding mutants would also be enhanced.5.The above results suggest that high temperatures can gradually expand the spatial structure of HarpinX,but the HR activity of the protein is heat-resistant.It may be that high temperatures can not destroy the amino acid sequence of HarpinX,so the key amino acid of heptad in the sequence can still form CC through hydrophobic interaction.Therefore,after high temperature treatment,HarpinX can still interact with the interacting proteins in tobacco leaves,resulting in necrotic spots of HR.The preliminary study on the heat-resistant protein mechanism of HarpinX not only lays a foundation for further study on the mechanism of Harpin,but also provides a theoretical basis for reforming the industrial non-heat-resistant proteins.