Identification of the PcMPK3 gene promoter from sweet cherry rootstock and its response to plant pathogen infection

Abstract: 【Objective】PcMPK3 encoding a mitogen-activated protein kinase (MAPK) in sweet cherry dwarf rootstock Gisela 6 (P. cerasus × P. canescens) is highly identical to Arabidopsis AtMPK3 gene, which is involved in plant growth, development and responses to biotic and abiotic stresses. Our previous work showed PcMPK3 responded to osmotic, salt stresses and bacterium pathogen infection significantly on the transcriptional level. However, the upstream regulation sequence of PcMPK3 has not been reported to date. The purpose of this study is to identify the promoter sequence of PcMPK3 and investigate its transcriptional regulation characteristics in sweet cherry rootstock.【Methods】The 5'-flanking upstream region of PcMPK3 gene was cloned by thermal asymmetric interlaced PCR (TAIL-PCR) using a degenerate primer AP1 and three gene-specific primers SP1, SP2 and SP3. Sequence analysis was carried out by online bioinformatics resources, such as Neural Network Promotor Prediction, softberry, PLACE and PlantCARE to reveal its basic core promoter, transcription start site and cis-regulatory elements (CREs) . The PcMPK3 promoter sequence (designated as PcMPK3 pro) was cloned into the plant expression vector pBI121-SN1 to replace CaMV35 S promoter and produce a new recombinant expression vector named pBI121-PcMPK3:GUS. The binary plasmid was then transformed into Agrobacterium tumefaciens strain LBA4404. Tobacco leaves (Nicotiana tobacum cv. Samsun) were incubated at 25℃under a light intensity of 300 μmol·m^-2·s^-1 and 16 hours/8 hours light/dark cycles for 4 weeks and then used for Agrobacterium-mediated transient transformation. The tobaccos were classified as control and treatment groups. For the control group, the leaves were inoculated by the suspension of the Agrobacterium strain LBA4404 (OD600=0.1) containing pBI121-PcMPK3:GUS vector. For the treatment groups, the tobacco leaves were inoculated by the suspension mixture of Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) (OD600=0.01) and Agrobacterium LBA4404 (OD600=0.1) harboring pBI121-PcMPK3:GUS vector. Both suspensions for the two groups were prepared with the infiltration buffer containing 10 mmol·L^-1 MgCl2, 10 mmol·L^-1 2- (N-morpholino) ethanesulfonic acid (MES) and 150 μmol·L^-1 Acetosyingon and injected into the tobacco leaves by 1 mL syringe, respectively. The infiltrated leaves were harvested for GUS activity assay at 12 hours and 24 hours after infiltration. For the GUS activity assay, fresh leaves were subjected to X-Gluc solution for histochemical staining and were quantitatively measured using the fluorometric determination method.【Results】The first and second round TAIL-PCR products showed dispersive results during electrophoresis detection. For the third round PCR, two distinct fragments, more than 2 000 bp, were amplified and purified from gel for sequencing respectively. A sequence of 2 027 bp, located in the 5'-flanking upstream of PcMPK3 gene, was finally cloned and designated as‘PcMPK3 pro'. It was registered at GenBank as KY434098. Sequence analysis showed basic promoter may exist at the position of-123 bp to-172 bp, -1 652 bp to -1 702 bp and-1 795 bp to -1 845 bp in the upstream of ATG initiation codon. Typical TATA box and CAAT box elements and numbers of cisregulatory elements relating to plant defense responses were also contained within PcMPK3 pro, including the bacterium and fungal elicitor, abscisic acid, ethylene, salicylic acid (SA) , methyl jasmonate (MeJA) and gibberellin responsive elements. This means PcMPK3 pro may be controlled by phytohormones and involved in physiological growth and development during pathogen infection. Two MYB transcription factors binding sites were also contained within the promoter sequence, which meant MYB transcription factors family may take responsibility to regulate MAPK genes expression. In order to uncover further evidence of PcMPK3 pro characteristics, the recombinant plasmid pBI121-PcMPK3 pro:GUS was digested by Hind III and BamHI for vector construction determination. Different infiltration suspensions, either agrobacterium LBA4404 harboring pBI121-PcMPK3 pro:GUS vector or bacterium mixture with Pst DC3000 and LBA4404 (pBI121-PcMPK3 pro:GUS contained) were injected into tobacco leaves, respectively. For the histochemical assay, blue staining can be observed in both groups 12 hours after inoculation. However, the intensity of the treatment group was much higher than the control group, about4.42 times higher than that in control groups according to the fluorometric quantitative assay. More obvious differences were observed 24 hours after inoculation. This result indicated that the transcriptional expression of GUS gene can be driven by the promoter PcMPK3 pro and induced quickly in response to the challenge of plant pathogen.【Conclusion】Previous research showed most of the plant MAPK members can be regulated at transcriptional level during biotic or abiotic stresses challenging. However, more attentions were paid to phosphorylation of the kinase and their interaction elements in the downstream.Few study on the transcriptional regulation of plant MAPK genes was reported. Revealing the characteristics of MAPK genes' promoters and their CREs will provide valuable information for the cultivar improvement on sweet cherry rootstocks by genetic engineering methods. In this study, the PcMPK3 promoter was isolated and characterized. It contained typical TATA box and CAAT box elements and numbers of stress-related CREs and can drive the reporter genes quickly during Pst DC3000 infection. Therefore, PcMPK3 gene may be involved in plant physiological responses to pathogen challenging.