Cloning, Bioinformatic Analysis and Expression Pattern of Phospholipase D Gene Family in Vitis vinifera

Background: The phospholipase D genes have been identified to play critical roles in plant growth and stress responses. There are 12 and 17 PLD members conformed in Arabidopsis and Oryza sativa respectively. Different PLD isoforms have distinct regulatory and catalytic properties. Method: In this study, the VvPLD genes were cloned using the genome-wide search and RT-PCR amplification from suspenson-cultured grape cells (Vitis vinifera L. cv. Cabernet Sauvignon). A comprehensive bioinformatics analysis of the VvPLD family was then performed. To further investigate the function of the VvPLD genes during pathogen infection process, Botrytis cinerea was used to attack suspenson-cultured Cabernet Sauvignon cells. The mRNA expression patterns of VvPLDs were examined by quantitative real-time PCR. Results: Ten PLD coding sequences (CDS) and two PLD genes segments were isolated from grape berry suspension cells. The VvPLDs were characterized and classified into 6 types (2 VvPLD alpha s, 2 VvPLD beta s, 3 VvPLD delta s, 1 VvPLD epsilon, 1 VvPLD rho and 1 VvPLD zeta) and 3 groups (C2-PLD, PXPH-PLD and SP-PLD). Quantitative real-time RT-PCR analysis showed that VvPLD beta 1, VvPLD beta 2, VvPLD delta 2, VvPLD rho and VvPLD zeta were up-regulated, whereas VvPLD alpha and VvPLD delta were down-regulated during Botrytis cinerea infection. Immunoblotting with AtPLD alpha 1 antibodies detected a higher abundance of VvPLD alpha in infected grape cells, which was in accordance with its enzyme activity. Conclusion: The results of this study will be useful in selecting candidate genes related to disease resistance in grapevine and pave the way for further functional verification of the VvPLD genes.