Cloning And Functional Analysis Of Wheat Alkalin/neutral Invertase (Tα-A/N-Inv) Genes Induced By Puccinia Striiformis F.sp.Tritici

The plant pathogenic fungi are the most economically important plant pathogen.Wheat stripe rust,caused by Puccinia striiformis f.sp.tritici,is considered one of the major wheat disease in China.The epidemics of the disease often caused tremendous losses of wheat yield in China.So far,over 40 stripe rust resistance genes have been identified in world,and it is of great important in understanding the molecular mechanism of wheat-stripe rust interaction in order to prevent and control wheat stripe rust.The cloning of disease resistance genes and the dissection of the disease resistance related signal pathways will provide insights into the mechanism of host-pathogen interaction and theoretical basis for the sustainable control of stripe rust.In this study,a Ta-A/N-Inv was characterized in the wheat-stripe rust fungus pathosystem.The main results and conclusions are as follows:We search the wheat cDNA libraries constructed in our lab and obtain a 1659bp coding sequence,which contained an open reading frame(ORF)encoding a polypeptide of 552 amino acids with a molecular weight of 62.89 kDa and pI of 6.1.The protein conserved domain analysis indicated that the primary structure of Ta-A/N-Inv was composed of a six-hairpin glycosidase-like domain and a glycosyl hydrolase family 100 domain(GH100).GH100 was conserved domain of A/N-Inv.The phylogenetic analysis of Ta-A/N-Inv and other Inv revealed that Ta-A/N-Inv was most similar to Oryza sativa,Zea mays and Setaria italica.To test the subcellular localization of Ta-A/N-Inv,we carried out transient expression analysis of Ta-A/N-Inv-GFP fusion protein in Arabidopsis leaf protoplasts and found that Ta-A/N-Inv is probably a cytosolic protein.Heterologous expression of Ta-A/N-Inv in S.cerevisiae,suggested that Ta-A/N-Inv was an A-Inv.To examine the impacts of stripe rust on the total alkaline/neutral invertase,soluble invertase activities were measured quantitatively,and an increase in the activity of A/N-Inv was detected.The quantitative real-time polymerase chain reaction(qRT-PCR)analyses revealed Ta-A/N-Inv abundance in green leaf.Both cold and wound treatment induced a decrease in transcript level from 2h to 6h but an increase from 12h post treatment.The down-regulation of the Ta-A/N-Inv transcript was observed on exogenous application of JA,SA,ABA and ET immediately following treatment from 2h post treatment.In wheat leaves challenged by the virulent Pst race CYR23,the level of the Ta-A/N-Inv transcription did not change significantly,but in the compatible interaction challenged by the virulent Pst race CYR31 increased as early as 12h post inoculation(hpi).To obtain direct evidence for the function of Ta-A/N-Inv during the wheat-Pst interaction,BSMV-VIGS system was used to decrease the transcript level of Ta-A/N-Inv,which enhanced the resistance of wheat to the virulent race CYR31.The results indicated that Ta-A/N-Inv may involve in wheat defense responses to stripe rust through change the reactive oxygen species homeostasis.