Isolation,Identification And Functional Analysis Of Oxalate-Binding Proteins In Arabidopsis Thaliana

Sclerotinia sclerotiorum(Lib.) de Bary is a devastating and cosmopolitan plant pathogen causing sclerotia stem rot on many crops such as oilseed rape and lettuce.Oxalic acid is considered to be one of the most important pathogenic factors in Sclerotinia sclerotiorum,but the accurate targets are still unclear.Studies of the action mode of oxalic acid and exploring the oxalate-binding proteins in plants will be helpful for elucidating the targets of oxalic acid in plants,and provide new clues for subsequently control of Sclerotinia sclerotiorum.Therefore,the following research was carried out:The effects of different oxalic acid with different pH value and concentration on the recovery of pathogenicity of Sclerotinia sclerotiorum strain Ep-1PB were determined,and the accumulation of oxalate under different pH value in Nicotiana benthamiana protoplasts was studied.The results showed that the exogenous addition of oxalic acid could partially restore the pathogenicity of Ep-1PB strain,and the effect was significant than citric acid.The concentration of oxalic acid was positively correlated with the pathogenicity of Ep-1PB strain in a certain concentration range.In addition,the lower of pH value,the stronger rescovery of pathogenicity of strain Ep-1PB.Under acidic conditions,the amount of oxalate detected in Nicotiana benthamiana protoplasts was significantly higher than that in neutral and alkaline conditions.A method for isolation of oxalate-binding proteins in Arabidopsis thaliana was developed,including optimizing the method for construction of oxalic acid-containing gel beads and using the system for isolating oxalic acid binding proteins.The results showed that the pH value of 5.6-6.0 was optimum for the reaction system.Adding the oxalic acid to adjust the pH to 6.0 after 1 h of the reaction could improve the reaction efficiency,and ddH2 O was the best solvent.A total number of 79 proteins were indentified as oxalate-binding proteins in Arabidopsis thaliana.GO analysis of these proteins showed that they were involved in antioxidant stress,gene silencing mediated by RNA,cell response to light intensity,cellular response to disaccharide,and cellular response to sucrose stimulation and other biological processes.The GO enrichment analysis showed that the laregest protein group was involved in anti-oxidative stress,including anti-superoxide,superoxide metabolism and removal of superoxide ion.Meanwhile,the KEGG analysis showed that these oxalate-binding proteins were also involved in the pathway of amino acid synthesis,carbon metabolism,tricarboxylic acid cycle,and carbon fixation of photosynthesis.KEGG enrichment analysis indicated that the most significant enriched pathway was the synthesis of phenylpropanone,starch and sucrose metabolism.The innovations of this study include:(1)It uncovered that H~+ could enhance the pathogenicity of Sclerotinia sclerotiorum by promoting the entry of oxalates into plant cells;(2)constructed a method for isolating plant oxalate-binding proteins;(3)the oxalate-binding proteins in the planst were isolated,identified and bioinformatically analyzed for the first time,which provided a new sight for the further study of the target of oxalic acid in plants.