Functional Analysis Of TaSWEET14 Gene During Wheat And Puccinia Striiformis F.sp Tritici Interaction

Wheat stripe rust,caused by an obligate fungus Puccinia striiformis f.sp tritici(Pst),is one of the important epidemic disease of wheat in China.Large scale outbreak of wheat stripe rust often result in a significant decline in production,sometimes even crop failure.Being an obligate parasitic pathogen,the nutrition of Pst is mainly from the host plant after infection,in which process the sugar transporter plays an important role.To study how the mechanism of the wheat SWEET sugar transporter supply nutrition to the pathogen,we screened wheat SWEET gene which induced by Pst from the SWEET family by using the method of biochemistry and molecular biology.Then we analysed the characteristics of the gene and the preliminary function in the interaction of the wheat and the Pst.The main results are as follows:(1)We searched the wheat cDNA libraries constructed in our lab and obtained 13 wheat SWEET(TaSWEET)gene which has a complete ORF sequence by using bioinformatics.Phylogenetic analysis of TaSWEET revealed that TaSWEET was most similar to rice SWEET.(2)We obtain 9 TaSWEET gene using the wheat leaf cDNA template by the technology of RT-PCR.(3)Analysed the expression pattern of the 9 genes by qRT-PCR and the result showed that the TaSWEET14 gene is induced by Pst,TaSWEET14 transcript was observed in the compatible interaction challenged by the virulent Pst race CYR31 increased as early as 72 h post inoculation(hpi).(4)Analysis of its tissue specificity found that TaSWEET14 were expressed in many organizations,and we found that the expression is the lowest in leaf,but the expression is the highest in the shell.In abiotic stress(salt,drought,low temperature,injury),the expression of TaSWEET14 is significantly induced in NaCl and PEG treatment after 48 h;In plant hormone treatment(ET,ABA and SA,MeJA),an up-regulation of TaSWEET14 transcript was observed,and we found ABA and ETH is higher than others.(5)The gene TaSWEET14 we obtained has 891 bp coding sequence,which contained an open reading frame(ORF)encoding a polypeptide of 297 amino acids with a molecular weight of32.38 kDa and pI of 8.98.Analysising the genetic structure domain of TaSWEET14,we found TaSWEET14 contains seven transmembrane domains.(6)Heterologous expression ofTaSWEET14 in S.cerevisiae mutant strain suggested that TaSWEET14 has the ability to transport glucose,fructose,mannose and sucrose,which proved that TaSWEET14 was an sugar transporter.To test the subcellular localization of TaSWEET14,we carried out transient expression analysis of TaSWEET14-GFP fusion protein by injection the tobacco and found that TaSWEET14 is probably a membrane protein.We found that TaSWEET14 was the form of a dimer when functionating by using the method of bimolecular fluorescence complementation.(7)To obtain direct evidence for the function of TaSWEET14 during the wheat-Pst interaction,BSMV-VIGS system was used to decrease the transcript level of TaSWEET14,which restricted the expansion of the hyphae,showing that the colonies area is reduced obviously in the compatible.From the above,Pst obtain sugar from host cell through regulating the expression of the host TaSWEET14 gene,then promote the pathogen to infection.