Molecular characteristics and biochemical functions of VpPR10s from Vitis pseudoreticulata associated with biotic and abiotic stresses

Grapes are one of the world's oldest and most important fruit crops. They are of high economic value in many countries, but the susceptibility of the dominant winegrape species Vitis vinifera to fungal disease is a significant problem. The Chinese wild grape species are a rich source of disease-resistance genes and these can be used to discover how disease resistance in V. vinifera grapevines might be enhanced. Pathogenesis-related (PR) 10 proteins are involved in the disease-response. Here, we use the genomic DNA of the Chinese wild species Vitis pseudoreticulata accession "Baihe-35-1" as the template to design specific primers based on VvPR10s sequences. We used overlap extension PCR to obtain the sequences: VpPR10.4, VpPR10.6, VpPR10.7 and VpPR10.9. The coding sequences of the VpPR10s were then cloned into the pGEX-4T-1 vector. The purified proteins VpPR10.4, VpPR10.6, VpPR10.7 and VpPR10.9 were used to analyse nuclease activity. Meanwhile, functional analysis of VpPR10s under different biotic and abiotic stresses was carried out to further clarify the disease-resistance mechanisms of the Chinese wild grapevine VpPR10 genes. The analysis of protein structure indicates that VpPR10.4 and VpPR10.7 had the P-loop domain and the Bet v 1 motif, which are a consistent feature of plant PR10. However, there was no P-loop domain or Bet v 1 motif in VpPR10.9 and we could not find the Bet v 1 motif in VpPR10.6. The results of the nuclease activity assay and of the functional analyses of VpPR10s under different biotic and abiotic stresses also confirm that VpPR10.4 and VpPR10.7 proteins have marked RNase, DNase, anti-fungal activities and respond to abiotic stresses. The VpPR10.6 and VpPR10.9 proteins do not have these activities and functions.