Function Study Of Soybean GmSWEET15/31 Genes In The Interactions Of Sclerotinia Sclerotiorum With Its Host Plants

Sclerotinia sclerotiorum（Lib.）de Bary is a plant pathogenic fungus that seriously harms many crops such as soybean,rape,sunflower and vegetable,which causes serious economic losses worldwide every year.Sclerotinia stem rot of soybean caused by S.sclerotinia is a serious fungal disease,which is very difficult to control.In epidemic years,the yield loss of soybean can reach 20 % ～ 30 %,in severe plots,it can reach 50 %,and even lead to the total yield loss,which seriously affects the yield and quality of soybean.SWEETs（Sugars Will Eventually Be Exported Transporters）is a member of the sugars family,which plays an important role in the growth and development of plants.It includes carbohydrate transport,phloem loading and unloading,growth and development,abiotic stress and plant-pathogen interaction.Current studies on SWEET proteins in host-pathogen interactions mechanisms are mainly focused on Arabidopsis and rice,and less on other plants,especially in soybean and fungal interactions.To reveal the mechanism of the role of soybean GmSWEETs proteins in the interaction between S.sclerotinia and soybean,this study revealed that soybean SWEET proteins were induced to be expressed by S.sclerotinia infestation by analyzing transcriptomic data from S.sclerotinia infested soybean.On this basis,the soybean GmSWEET15/31 genes was used to transform the model plant Arabidopsis for heterologous expression of GmSWEET15/31,along with a T-DNA insertion mutant and a CRISPR-edited mutant corresponding to the homologous genes At SWEET12/1 in Arabidopsis,We observed and analyzed the mutant interactions with S.sclerotinia,determined the changes in sugars,and bound the expression of PR protein-related genes and defense enzymes to clarify the function of soybean SWEET protein in the interactions between S.sclerotinia and the host,and further elucidate the molecular mechanism of SWEET protein regulation of plant immunity.The main research results are as follows:1.The expression of soybean GmSWEET15/31 genes was determined by q RT-PCR after inoculation with S.sclerotinia.The results showed that both genes were downregulated and their biological properties were analysed by cloning and using bioinformatics software.Bunsen burnet transiently expressed GmSWEET15/31 and subcellular localization showed that both were localized to the cell membrane.2.In order to clarify the function of GmSWEET15/31 proteins in the S.sclerotinia and host interaction,three plants of Arabidopsis overexpressing GmSWEET15 and GmSWEET31 genes,OE-GmSWEET15#1,OE-GmSWEET15#2,OE-GmSWEET15#3and OE-GmSWEET31#1,OE-GmSWEET31#2,OE-GmSWEET31#3,were obtained by flower dip method in this study.After infection of wild-type（Col-0）and transgenic Arabidopsis by S.sclerotinia,symptom phenotypes were observed and spot areas were counted.Disease symptoms were reduced in transgenic GmSWEET15/31 Arabidopsis compared to the wild type,and the biomass of S.sclerotinia in transgenic Arabidopsis was found to be lower than that of the wild type as determined by q RT-PCR.The glucose and sucrose contents of the plants were measured before and after the infestation and the results showed that the glucose and sucrose contents of the transgenic plants not inoculated with S.sclerotinia were the same as those of the wild type under the same culture conditions.both glucose and sucrose content increased after inoculation with S.sclerotinia,and the up-regulation was more pronounced in the wild type,with non-significant differences.The glucose content of adjacent leaves of the same plant inoculated with S.sclerotinia was close to that before inoculation,and the growth rate of S.sclerotinia was faster in the medium containing glucose.For defenserelated index measurements,it was found that 18 h after inoculation of plants with S.sclerotinia,the transgenic GmSWEET15/31 Arabidopsis had higher expression of salicylic acid pathway-related genes PR1 and PR2 and about 2-fold higher expression of NPR1 gene and lower expression of jasmonic acid pathway-related gene PDF1.2than the wild type.The activities of defense-related enzymes SOD,POD,and CAT were enhanced upon S.sclerotinia infestation and were lower in transgenic Arabidopsis than in wild type.3.To further investigate the mechanism of the role of GmSWEET15/31 protein in the S.sclerotinia and host interaction,Arabidopsis At SWEET12/1 was found to be a homolog of GmSWEET15/31,respectively,by homology comparison,and T-DNA insertion mutants and CRISPR-edited mutants of wild-type and At SWEET12/1 were used to infest the S.sclerotinia.The results showed that the Arabidopsis mutant had a larger lesion area than the wild type,the S.sclerotinia accumulated more in the mutant strain,and there were no significant differences in glucose and sucrose in the uninoculated mutant strain compared with the wild type.both glucose and sucrose increased in the inoculated leaves after S.sclerotinia infestation,and the sugar content was higher in the mutant than in the wild type,and for the inoculated adjacent leaves of the same plant,the sugar content was close to that before inoculation.Among defense-related genes,mutants At SWEET12/1 had lower PR1 and PR2 genes expression than wild type and higher PDF1.2 gene expression than wild type 18 h after inoculation of the plants by S.sclerotinia.The SOD,POD,and CAT enzyme activities of both wild type and mutant At SWEET12/1 were enhanced after S.sclerotinia infestation,and the mutant was higher than the wild type.In summary,GmSWEET15/31 exert disease resistance in the S.sclerotinia and host interaction and may affect host immunity by regulating sugar transport and the expression of related genes during invasion of host plants by S.sclerotinia.