Cloning Of Wheat Autophagy Related Genes And Functional Analysis Of TaATG6 And TaATG3 Genes

Wheat stripe rust, caused by Puccinia striiformis f.sp. tritici, is widely spread around the world. The pandemic of wheat stripe rust can cause severe yield reduction of wheat, selection and use of rust-resistant varieties are the most reliable method in controlling the disease. Hypersensitive response is a typical resistance reaction,of wheat after infected by stripe rust, studying the role and molecular mechanism of autophagy genes in HR-PCD is very important to reveal the interaction between wheat and stripe rust pathogenic mechanism.In this study, the analysis of Arabidopsis, Oryza Sativa and Brachypodium sylvaticum autophagy gene sequence, primers were designed according to conserved regions, the successful cloning of the six wheat autophagy-related gene, named TaATG8a, TaATG8c, TaATG8h, TaATG18c, TaATG3 and TaATG6.TaATG8a length 695bp, encoding 119 amino acids, TaATG8c length 647bp, encoding 119 amino acids, TaATG8h length 532bp, encoding 117 amino acid, sequence analysis showed that three encoded proteins has a class of conserved ubiquitin fold.TaATG8a and TaATG8h sharing high homology with Arabidopsis ATG8 protein as well as TaATG8c sharing high homology with corn ATG8 protein respectively.Three wheat Atg8 genes were induced by stripe rust in different levels. After inoculation of stripe rust TaATG8a expression was up-regulated in incompatible interaction and down-regulated compatible interaction. TaATG8c and TaATG8h expressions were both up-regulated in incompatible and compatible interactions. TaATG8a and TaATG8h transcripts in wheat leaves was induced by exogenous applied methyl jasmonate (MeJA), ABA and ethylene. However, salicylic acid (SA) had no obvious effect on the expressions. TaATG8c transcript is not sensitive to exogenous hormones, processed slightly fluctuate. TaATG8a and TaATG8h possible through mediated wheat stripe rust by the JA and ethylene signaling pathways in the defense response, salicylic acid and jasmonic acid are obvious antagonism in defense response of wheat stripe rust. Environmental stimuli, including drought, high salinity and wounding, also induced TaATG8a expression.while low-temperature lead reducesd TaATG8a expression.Under drought and high salinity environment, TaATG8c expression was down-regulated, while under wounding and low-temperature environment the expression of TaATG8c was up-regulated.; TaATG8h was very sensitive to environmental stress and always up-regulated in all treatments. The Atg8 members may separately or jointly involved in the different resilience mechanisms of wheat.TaATG18c length 1205bp, encoding 348 amino acids, sequence analysis showed that TaATG18c has multiple WD40 repeats sequences and contains three transmembrane domains.TaATG18c sharing high homology with Arabidopsis ATG18. Quantitative analysis showed that, TaATG18c transcripts was induced in compatible interaction. TaATG18c transcripts in wheat leaves was induced by exogenous applied methyl jasmonate (MeJA), ABA and ethylene. However, salicylic acid (SA) had no obvious effect. Environmental stimuli, including drought, high salinity and wounding, also induced TaATG18c expression.while low-temperature lead reducesd TaATG18c expression.TaATG18c may be involved in the nutrients transfer in wheat after the wheat stripe rust infection.TaATG3 length 954bp, encoding 317 amino acids; TaATG6 length 1556bp, encoding 504 amino acids, two encoded proteins both has a transmembrane helix region. TaATG3 sharing high homology with barley ATG3 and TaATG6 sharing high homology with rice ATG6. Quantitative result shows that, TaATG3 and TaATG6 gene transcription induced by wheat stripe rust, and the expression of incompatible interaction is much higher than the compatible interaction, they may be involved in the interaction between wheat and the stripe rust. After silencing wheat TaATG3 and TaATG6 genes by VIGS technology, it was found that of area of chlorosis was larger in wheat leaves and cell death had spread to healthy leaf tissue. Fluorescence staining showed increased number of dead cells points of transfected BSMV-ATG3 and BSMV-ATG6 leaves, also the necrosis area had expand, the length of stripe rust infection hyphae had slight increased, the expansion of stripe rust even more serious. TaATG3 and TaATG6 genes are likely involved in the hypersensitive cell death response in the inhibition of the process of pothogen expansion, and wheat disease resistance.