Characterisation of the Potyvirus VPg Interacting Protein (PVIP) gene from Nicotiana benthamiana
According to a study by Dunoyer et al (2004), a protein called Potyvirus VPg-Interacting Protein (PVIP) is known to interact with potyviruses upon infection of the host plant Arabidopsis thaliana and help in their replication and movement. Potyvirus are the largest group of plant pathogens causing severe damage to most crops. PVIP appears to be a plant specific protein that occurs in uninfected tissues, suggesting a role which is independent of viral infection. Potyviruses appear to highjack this protein for their own needs. What the function of PVIP in healthy cells is unclear. This study aimed to determine the function of PVIP in Nicotiana benthamiana. Bioinformatic analysis was used to identify sequence homologues of the N.benthamiana PVIP protein sequence, from all the plant species. Phylogenetic analysis of these homologues showed this to be an ancient gene, being present in moss, ferns, gymnosperms and angiosperms, and that there are likely to be two genes that have arisen via gene duplication. Only one gene from N.benthamiana has been identified, thus, there is likely to be another gene to be found. The function of many of these homologues, together with the identification of a PHD finger domain within PVIP from A.thaliana suggested a role for the protein in transcriptional regulation. The 'PHD finger' domain being plant specific; proteins with this domain are likely to have a role in a plant specific function. Analysis of the promoter of the PVIP gene may provide more insight into gene functionality and also the hidden transcriptional network of the gene. Bioinformatic analysis of the 5' flanking regions of the PVIP homologues from A.thaliana and Oryza sativa (rice), predicted that the PVIP gene would be responsive to changes in conditions of light and have a role in development. The closest homologues from A.thaliana, OBERON (OBE) 1 and 2 have been shown to functionally overlap with roles in apical and root meristem development/maintenance. The OBERON proteins also control the transcription of genes required for auxin responses through the action of their PHD finger domains. This suggests that the rice homologues on chromosomes 11 and 12, as well as the N. benthamiana proteins are functionally equivalent. Southern hybridisation and inverse polymerase chain reaction were used to isolate the 5' flanking region from the N.benthamiana PVIP gene. However, all attempts using either technique were unsuccessful. Quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) was used to study the relative amounts of PVIP mRNA in healthy tissues from N.benthamiana as well as how it responded to dark/light treatment. PVIP mRNA was shown to be expressed at the mRNA level in leaf, stem, root and flower tissue to varying degrees. The mRNA was most abundant in stem and least abundant in roots. The PVIP gene was predicted to be light responsive since potential binding sites for light responsive transcription factors were identified within the 5' flanking regions of the A.thaliana and rice homologous genes. Leaf tissue from N.benthamiana exposed to continuous dark for 48 hours was shown to have 0.3 to 3 fold change of PVIP mRNA compared with leaf exposed to continuous cycles of of 16 hours light / 8 hours dark. Leaf that had been exposed to the dark for 24 hours and then returned to 16 hours light/8 hours dark for one cycle showed lower levels of PVIP mRNA than leaf continuously exposed to the standard day/night cycles. These findings suggest that PVIP is indeed responsive to changes in light conditions. PVIP has previously been identified as a protein that interacts with potyviruses and has been implicated as having a role in viral movement through an infected plant. It's role in healthy tissue in unknown. However, bioinformatic analysis suggested that it has a role in transcription and it is likely to be developmentally and/or light regulated. Responsiveness to light was confirmed by RT-qPCR as was its expression in several tissues. This study suggests it has a role in flowering and shoot and root induction. It may also prevent pathogen attack and bring about DNA repair.