Abstract:

Objective: SPL (SQUAMOSA promoter-binding protein-like) is a class of plant specific transcription factors. They play important roles in plant growth and innate immune responses. Studies that focused on dissecting SPL gene function in poplar by using genetic transformation are still very limited. In order to explore the biological function of PdbSPL28 and its effects on disease resistance, the gene was overexpressed in Populus davidiana× P. bolleana, and differences in disease resistance between wild type (WT) and transgenic poplar were investigated. Results of this study would provide a basis for comprehensive understanding of the molecular mechanism by which the PdbSPL28 regulates disease resistance in poplar. Method: The full-length ORF of P. davidiana× P. bolleana PdbSPL28 was obtained by using homologous cloning method, and the miR156 binding site within the ORF was modified via artificial DNA synthesis technology. The pre- and post-modified PdbSPL28 were separately used to construct overexpression vector through enzymatic cleavage and ligation reactions. A vector was constructed by using the Gateway strategy, transiently transformed with tobacco epidermal cells, and used for sub-cellular localization analysis of PdbSPL28 protein. PdbSPL28 overexpression transgenic P. davidiana× P. bolleana was obtained with the method of agrobacteria-mediated leaf disc transformation, and the differences between WT and transgenic P. davidiana× P. bolleana in their resistance, leaf structure and disease index to two Diaporthe pathogens (Diaporthe nobilis and D. cercidis) were investigated with the combination of stab-injured inoculation and paraffin section technique. The urediniospore suspension of D. nobilis was sprayed on detached leaves of WT and transgenic P. davidiana × P. bolleana, and the leaves were sampled at different time after inoculation. The variations of jasmonic acid (JA) content were measured using liquid chromatography-mass spectrometry. Result: The ORF of PdbSPL28 is 1 341 bp in length and harbors one miR156 binding site. The PdbSPL28 protein has the conserved SBP domain and is localized in nuclear. Six PdbSPL28 overexpression transgenic lines were successfully obtained using the modified ORF within which the miR156 binding site was mutated. Compared with WT, transgenic P. davidiana× P. bolleana overexpressing PdbSPL28 was more resistant to two Diaporthe pathogens. Results of JA measurement indicated that the JA contents in the leaves of PdbSPL28 overexpression lines were higher than that of WT before inoculation. However, at 96 h after inoculation, the JA contents in the leaves of PdbSPL28 overexpression lines were significantly lower than that of WT. Conclusion: Sequence modification of miR156 action site can improve the probability of obtaining PdbSPL28 gene overexpression transgenic P. davidiana× P. bolleana. Overexpression of PdbSPL28 gene can enhance the resistance of P. davidiana× P. bolleana to leaf spot disease caused by Diaporthe pathogens. JA signaling pathway may play an important role in regulating the defense responses of overexpression lines during the D. nobilis infection. Results of this study forms a basis for further revealing the molecular mechanism of resistance to Diaporthe pathogens regulated by PdbSPL28 in P. davidiana× P. bolleana.

Key words: Populus davidiana× P. bolleana, SPL28 gene, overexpression, leaf spot disease, Diaporthe