Functional analysis of VdMAPK7 from Chinese wild spine grape (Vitis davidii Föex) in response to Colletotrichum viniferum

Abstract:【Objective】Mitogen activated protein kinase (MAPK) cascade pathway, a major signal transduction pathway widely distributed in eukaryotes, has an important function in plant responses to various biotic and abiotic stresses. Transcriptome analysis of Chinese wild spine grape (Vitis davidii Föex) infected by Colletotrichum viniferum was performed in a previous study. The infected grape fruits at 7 dpi showed significant changes in gene expression, and that the up-regulated genes were en-riched for those MAPK cascade, calcium ion binding and serine/threonine kinase. The VdMAPK7, se-lected from transcriptome data, showed highly up-regulated expression during C. viniferum infection, in-dicating that the VdMAPK7 was responsible for the resistance to C. viniferum. The main objective of this study was to clone and verify the function of the VdMAPK7 in response to C. viniferum.【Methods】Chinese wild spine grape (V. davidii ‘Fu’an’) was used as the experimental materials and the Vd-MAPK7 was selected based on the transcriptome data for further study. The grape berries were inoculat-ed by C. viniferum (strain FJ017) then the exocarps were collected after inoculation at different infection time points (0 d, 1 d, 3 d and 7 d) for further analysis. The RNA from C. viniferum infected grape exocarp were extracted by lithium chloride precipitation method and reverse transcription and cDNA  synthesis were performed using PrimeScript TM RT kit with gDNA Eraser. The expression patterns of the VdMAPK7 at different time points after C. viniferum infection were analyzed by qRT-PCR using GAP-DH (CB973647) as reference gene with 2^-ΔΔCt method. CDS sequence of the VdMAPK7 were cloned and the amino acid sequence of the VdMAPK7 was compared based on NCBI database, then other related proteins with high homology were screened. The phylogenetic relationship between the VdMAPK7 pro-tein and other related proteins were analyzed by cluster analysis using Phylogeny.fr platform. The full-length CDS sequence of the VdMAPK7 was obtained by sequencing, and the amino acid sequence of the VdMAPK7 was compared based on NCBI database. Sequence multiple alignment was carried out using DNAMAN (v6.0) and demonstrated with GenDoc v2.7.0. The VdMAPK7 was cloned into pCam-bia2300 vector and introduced into Agrobacterium tumefaciens GV3101, then the VdMAPK7 was tran-siently expressed in the tobacco leaves for subcellular localization and finally observed in confocal laser endomicroscopy (Leica TCS SP8) with excitation at 488 nm to elucidate the specific location in the cells. To study the response of the VdMAPK7 against C. viniferum, transgenic tomato plants of the Vd-MAPK7 were obtained using leaf disc transformation and inoculation assay of C. acutatum in the transgenic tomato of VdMAPK7 were carried out. The transgenic tomatoes inoculated by C. acutatum were collected after inoculation at different infection time points (0 h, 6 h, 12 h, 24 h, 48 h, 72 h), RNA ex-traction and cDNA synthesis were performed as described above. The expression patterns of the resis-tance genes (SlPR1 and SlPR2) were analyzed by qRT-PCR using SlActin as reference gene.【Results】Chromosomal location of the VdMAPK7 analysis was performed using the Blat- Search option. Vd-MAPK7 could be mapped on chromosome 4 of the European grape reference genome, which was dis-tributed in the region 18 972 351-18 978 329 bp. The amino acid homology identity of the VdMAPK7 cloned in this study compared with the VvMAPK7 (V. vinifera) was 99.18%. The results showed that the VdMAPK7 and the MAPKs from other species all contained S_TKc domain (32-319 aa). The phy-logenetic tree analysis showed that the VdMAPK7 was clustered with homologous proteins from V. vinifera, Solanum lycopersicum, S. tuberosum, Camellia sinensis, Davidia involucrata and Nicotiana tabacum. The results from qRT-PCR showed that the VdMAPK7 could response to the induction of C. viniferum, and highly expressed at the 7 days after inoculation. The results indicated that the VdMAPK7 could be induced to express in response to C. viniferum. The activated Agrobacterium solution containing the VdMAPK7 was transiently expressed into the N. benthamiana leaves and the fluorescence distri-bution was observed under a laser confocal microscope. The subcellular localization assay showed that the VdMAPK7 was localized in cytoplasm and nucleus. The transgenic tomato plants of the VdMAPK7 were obtained by leaf disk transformation method, and PCR detection was carried out using gDNA from transgenic plant leaves. Then, RNA from candidate plants (#2, #3, #9, #14, #21) was extracted and reverse transcribed, specific primers were designed according to the sequence of the VdMAPK7, and semi-quantitative PCR detection was carried out with SlActin as an internal reference, #2 and #9 plants were finally identified and selected for further study. Compared with the wild-type tomatoes, the Vd-MAPK7-overexpressing plants were shorter, with curled leaves and smaller fruits. To further verify the difference between the wild-type tomatoes and the VdMAPK7-overexpressed tomatoes, the single fruit weight, transverse diameter and longitudinal diameter of tomato fruit were measured in the study. The VdMAPK7 transgenic tomato fruits and the wild-type tomato fruits were inoculated with C. acutatum and the results showed that the over-expressed VdMAPK7 lines enhanced the resistance to C. acutatum. The results of qRT-PCR showed that SlPR1 and SlPR2 in response to salicylic acid signaling pathway were up-regulated in the VdMAPK7 over expressed tomato fruits inoculated with C. acutatum. The re-sults showed that the VdMAPK7 could enhance the resistance to C. acutatum in the transgenic tomatoes.【Conclusion】The VdMAPK7 could enhance the resistance of grape to C. viniferum and these results would provide theoretic basis for molecular breeding of spine grape for the resistance to C. viniferum.