Isolation And Characterization Of A Populus Trichocarpa PtrWRKY73 Transcription Factor On Resistance Of Disease And Drought In Transgenic Arabidopsis Thaliana

Populus as an economic and ecological importance of forest tree species, were often used in papermaking, rubber production and furniture manufacturing. However, due to their long life span, Populus often suffered from biotic and abiotic stresses. Plants were constantly challenged by drought and a variety of microbial pathogens like fungi and bacteria. Complex defense mechanisms have evolved in plants to protect themselves against the attack of stresses. Upon pathogen infection, plants rapidly activated defense responses mediating by multiple signal transduction pathways. Therefore, elucidation of the complex regulatory mechanisms for controlling defense gene expression among plant species was important to understanding the molecular basis of plant-pathogen interactions. WRKY transcription factors have been extensively studied of regulating genes associating with plant defense responses. This gene family had a lineage-specific expansion during the course of plant evolution, and consisted of approximately 100 members in Populus. In a previous study, we found several defense-related cis-elements distributed in the promoters of Poplar WRKY genes and about 60 WRKY genes were induced or down-regulated by treatments of SA, MeJA and Marssonina brunnea. However, the functional characterization of these WRKY genes in Populus, especially their roles in plant immune responses and drought responses, were limited.This study isolated PtrWRKY73 gene, which was induced by jasmonic acid methyl ester (MeJA), salicylic acid (SA), canker (Dothiorella greariasacc Sacc.,), sodium chloride (NaCl) and polyethylene glycol 6000 (PEG-6000), from Populus trichocarpa. PtrWRKY73 contained two WRKY domain, belonging to the WRKY I family, at the same time, in the N terminal had D domain and SP cluster, suggesting that PtrWRKY73 may be involved in the protein phosphorylation. Tissue expression spectrum analysis showed that PtrWRKY73 mainly expressed in root, old leaves, buds, and stem, particularly in the phloem in higher quantity. Subcellular localization experiment showed PtrWRKY73 protein accumulated in the nucleus, yeast single hybridization experiment proved that PtrWRKY73 proteins, which could activate the report gene, had the transcriptional activation activity. These results showed that the PtrWRKY73 protein was a transcriptional activation factor. In the overexpresion PtrWRKY73 Arabidopsis thaliana, disease-resistant analysis showed that the resistance of transgenic plants to the biotrophic bacteria Pseudomonas syringae pv. Tomato (Pst) DC3000 has been significantly improved, but to the necrotrophic fungi (Botrytis cinerea) the sensitivity was enhanced. At the molecular level, in the transgenic Arabidopsis, the SA pasway mediated disease resistance genes(PR1, PR2 and PAD4) increased significantly. The above results showed that the PtrWRKY73 positively regulated the SA mediated disease resistance, enhancing the plants to resistance to biotrophic pathogenic bacteria, but increasing the sensitivity to the necrotrophic pathogenic fungi.Meanwhile, over-expression PtrWRKY73 Arabidopsis was more resisitant to drought than wild type. Further, width to length ratio of the stomatal of wild type and transgenic plants L47 and L49 were 0.295131,0.141165 and 0.182188, respectively. The number of stomata per unit area of the wild type and transgenic plants L47、L49 were 175.774,158.3892 and 137.6774, respectively. These dates showed that not only the stomata’s width to length ratio of transgenic plants was less than the wild type, but also the number of per unit area was significantly less than the wild type. The results above indicated that PtrWRKY73 by reducing the size and the number of transgenic Arabidopsis stomata to enhance the drought resistance of transgenic plants. At the molecular level tested drought resistance related genes in the transgenic plants, finding that stress related marker genes RD29A, key enzyme of ABA synthesis pathway genes NCED3 and dehydration protein-coding genes RAB18 were significantly raised.The results above showed that PtrWRKY73 by positively regulating SA pathways to enhance the resistance of plants to biotrophic pathogens, but increase its sensitivity to the necrotrophic pathogenic fungi. On the other hand, PtrWRKY73 by controlling the size and number of stomata and promoting the expression of downstream genes of ABA passway to improve the ability of drought resistance in plants.