Research On The Expression Analysis Of β-1,3-glucanases In The Process Of Wheat And Leaf Rust Interaction

Our laboratory has long been engaged in the research on the physiological mechanisms of wheat Resistance to Wheat Leaf Rust Infection. Preliminary works have demonstrated that, in the incompatible wheat-leaf rust (Puccinia triticina) combinations,β-1,3-glucanase (β-1,3-glucanase, BG) has an important role in the resistance response induced by wheat leaf rust .In this experiment, wheat cultivar Lovrin10 and leaf rust fungus races 260 and 165 were defined as study object. Loverin 10 and 165 constitute the compatible combination , Loverin 10 and 260 constitute the incompatible combination. Systematic study ofβ-1,3-glucanase gene expression characteristics in both conbinations were carried out. Total RNA was extracted in both conbinations at 0h, 4h, 12h, 24h, 48h, 72h, 120h after wheat leaf rust infection andβ-1,3-glucanase gene expression characteristics were get by semi-quantitatively PCR after RT-PCR.The results showed that in both conbinations leaf rust infection could induceβ-1,3-glucanase gene expression, and the gene expression level in incompatible combination is higher than that in compatible combination. At 4h after inoculation,β-1,3-glucanase gene expression in the two combinations has showed a significant difference ,which may be related to host early resistance response .At 12h, 24h and 48h,β-1,3-glucanase gene expression has raised in both combinations, but the increase ofβ-1,3-glucanase gene in incompatible combination was significantly higher than compatible combination.At 72h and 120h,β-1,3-glucanase gene expression in incompatible combination continued to rise, however in compatible combinationβ-1,3-glucanase gene expression showed a downward trend.From the genetic level point of view, it is speculated thatβ-1,3-glucanase play an important role in the resistance response to wheat leaf infetion.To further demonstrate the reasons of the existence of these differences,it is studied from the protein level and physiological levels ofβ-1,3-glucanase which play a role in wheat resistance response induced by leaf rust fungus. Total protein was extracted in both combinations at above-mentioned time, andβ-1,3-glucanase were detected by western-blotting.The results showed thatβ-1,3-glucanase protein was detected at 4h after inoculation and gradually increased in both combinations. And the expression level in incompatible combination is higher than in compatible combination. In compatible combination enzyme expression levels began to reduce after 48 h, but in incompatible combinations the expression level maintained a high level. Then the enzyme activity in the bacteria leaves was detected and the activity trend is the same as the trend of the enzyme expression levels .All the results indicate thatβ-1,3-glucanase plays an important role in the wheat resistance response to leaf rust infection.Thenβ-1,3-glucanase spatial and temporal expression characteristics were studied in situ levels after leaf rust infection by immunolocalization.The results showed that:β-1,3-glucanase was mainly localized in the host and the host cell wall and cell gap solution and protein level in incompatibal combination is higher than in compatible combination, and this trend is more obvious in the later time. Resluts above prove thatβ-1,3-glucanase participates in the process of wheat resistance response to leaf rust infection and directly inhibitates the leaf rust infection.In summary, wheat varieties Loverin 10 and leaf rust physiological races 260 and 165 were chosen to form compatible and incompatible combinations. By semi-quantitative PCR, Western Blotting detection, enzyme activity assay and immuno-gold labeling technique at the transcriptional level, translation level, enzyme activity and in situ levels to studyβ-1,3-glucanase after leaf rust infection.The results have proved thatβ-1,3-glucanase plays an important role in the resistance responses induced by wheat leaf rust .The results lay a foundation for further study of physiological mechanisms of wheat resistance to leaf rust, and has an important role in our in-depth understanding of wheat defense mechanism to leaf rust.