| Wheat stripe rust caused by Puccinia striiformis f. sp. tritici(Pst) is one of the most serious fungal plant diseases threatening wheat production every year in China. Breeding and planting wheat cultivars that have resistance toward stripe rust are the most ecomonic, efficient and safty strategies. Hence, cloning plant defense-related genes in rust resistance pathways, as well as detailed characterization and annotation their functions provide strong supports for the selection and genetic improvement for the wheat breeding. Using updated biological techniques such as bio-informatic analysis, qRT-PCR, bombardment transient expression system, virus-induced gene silencing, yeast two-hybrid, bimolecular fluorescence complementation and immuno-localization assays, functional characterization of wheat program cell death-related Metacaspase gene TaMCA4and vesicle transportation-related SNAREs during wheat-Puccinia striiformis f. sp. tritici interaction has been achieved. In details:1. Programmed cell death (PCD) is a physiological process to remove redundant or harmful cells, for the development of multi-cellular organisms, or for restricting the spread of pathogens (hypersensitive response). Metacaspase are cysteine-dependent proteases, which play an essential role in PCD. TaMCA4is a Type II metacaspase gene cloned from wheat cultivar Suwonl1, with typical structural features such as peptidase C14caspase domain and a long linker sequence between the two subunits. Transient expression of TaMCA4in tobacco leaves failed to induce PCD directly but enhanced cell death triggered by a mouse Bax gene or a candidate effector gene from Puccinia striiformis f. sp. tritici(Pst). Enhancement of PCD was also observed in wheat leaves co-bombarded with TaMCA4. When challenged with the avirulent race of Pst, the expression level of TaMCA4in wheat leaves was significantly up regulated, whereas the transcript level was not induced by the virulent race. Moreover, knocking down TaMCA4expression by virus-induced gene silencing enhanced the susceptibility of wheat cultivar Suwonl1to the avirulent race of Pst and reduced the necrotic area at infection sites.2. Sub-cellular localisation of SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) and their ability to form SNARE complex are critical to determine the specificity of vesicle fusion. NPSN11, a member of Novel Plant SNARE (NPSN) genes, has been reported to be involved in the delivery of cell wall precursors to the newly formed cell plate during cytokinesis. However, functions of NPSN genes in plant-pathogen interactions are largely unkown. In this study, we cloned and characterised three NPSN genes (TaNPSN11, TaNPSN12and TaNPSN13) and three plant defense-related SNARE homologues (TaSFP132, TaSNAP34and TaMEMB-12). TaSYP132showed a highly specific interaction with TaNPSN11in both yeast two-hybrid and bimolecular fluorescence complementation (BiFC) assays. We hypothesize that this interaction may indicate a partnership in vesicle trafficking. Expressions of the three TaNPSNs and TaSYP132were differentially induced in wheat leaves when challenged by Puccinia striiformis f. sp. tritici(Pst). In virus-induced gene silencing (VIGS) assay, resistance of wheat cultivar Xingzi9104to the Pst avirulent race CYR23was reduced by knocking down TaNPSN11, TaNPSN13and TaSFP132, but not TaNPSN12, implying diversified functions of these wheat SNARE homologues in prevention of Pst infection and hyphal elongation. Immuno-localization results showed that TaNPSN11or its structural homologues were mainly distributed in vesicle structures near cell membrane toward Pst hypha. Taken together, our data suggests a role of TaNPSN11in vesicle-mediated resistance to stripe rust. |
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