Towards the analysis of Potyvirus VPg Interacting Protein (PVIP) gene expression in response to potyvirus infection
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Potyvirus is the largest genus in the RNA plant virus family of Potyviridae. Potyviruses infect most of the economically important agricultural and ornamental crops; Dasheen mosaic virus (DsMV) is a potyvirus which infects edible aroid plants such as taro, particularly in the South Pacific region. These viruses rely on various host plant proteins for their movement and replication. One such plant protein which is known to interact with the viral protein called virus genome linked protein (VPg) is potyvirus VPg interacting protein (PVIP). Dunoyer et al., (2004) suggested role for PVIP in potyvirus movement, in disease development and as an important factor for virus replication. Further, through bioinformatics sequence analysis of homologous Arabidopsis thaliana and Nicotiana benthamiana PVIP sequences, the PVIP gene was found to be homologous to OBERON 1 and OBERON 2 in A. thaliana, suggesting a role for PVIP in meristem maintenance (Saiga et al, 2008; Anand, 2010). This analysis also suggested that PVIP may have a role independent of virus infection, it may be an important factor in plant development. Anand (2010) analysed the effect of abiotic stress on PVIP mRNA accumulation. The PVIP mRNA levels were assessed in leaf tissue of N. benthamiana under various dark and light conditions. A decline in the PVIP mRNA accumulation was observed when these plants were placed in continuous dark, suggesting that light induces the expression of PVIP mRNA. This study concluded that PVIP gene is responsive to this type of abiotic stress; however, the responsiveness of PVIP mRNA level to biotic stress such as virus infection is yet to be unravelled. The aim of this study was to determine the variation of PVIP mRNA accumulation in healthy and DsMV infected taro using reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). To conduct this analysis, the first objective was to identify appropriate reference genes for use in virus infected studies in taro. There are several methods to conduct gene quantification studies such as northern hybridization, microarray data analysis; among these RT-qPCR has become the most reliable, common and sensitive method for the quantification of gene expression. According to the MIQE guidelines, there are many factors that need to be considered while conducting RT-qPCR analysis, one such important variable is the use of appropriate reference genes (Bustin et al., 2010). A reference gene is defined as having a stable expression under different experimental treatments (Taylor et al., 2011). Housekeeping genes such as glyceraldehydes-3-phosphate dehdrogenase (GAPDH), ubiquitin (UBQ), actin 18S rRNA have been used as reference genes; however, it has been found that the expression level of many of these housekeeping genes vary under various experimental conditions. Lilly et al., (2011) assessed the stability of 12 candidate reference genes in virus infected A. thaliana. Among these 12 genes studied such as actin, UBQ, only four genes were shown to have stable expression. These four genes were namely, F-box family protein (F-box), SAND family protein, protodermal factor 2 (PDF2) and elongation factor α (EF1α). These genes were suggested to be suitable reference genes for analysis of all virus infected plants; however, this would need to be tested in other species before using them. Therefore, in this study the expression stability of these four reference genes namely, EF1α, F-box, SAND and PDF2 in healthy taro and N. benthamiana was assessed using RT-PCR. Various parameters such as annealing temperatures were tested using gradient PCR to optimise for efficient amplification of these genes in taro and N. benthamiana; however, efficient amplification was not achieved in taro and N. benthamiana using these primer pairs. Further, sequence analysis of homologous A. thaliana EF1α, PDF2, F-box and SAND genes against other publicly available monocot and dicot sequences, suggested significant variation within the primer target sequences, particularly in the 3’ end of the reverse primers. This may account for the inefficient amplification of these genes in taro and N. benthamiana. Therefore, new generic primers, specific to both monocot and dicot species were designed; however, due to limited availability of sequence information for PDF2 and SAND in publicly available databases along with significant variations among monocots and dicots, new generic primers were only designed for the amplification of EF1α and F-box genes. A EF1α primer pair suitable for both monocots and dicots was designed. For F-box, two primer pairs were designed one for monocots and one for dicots. The newly designed primers were then tested on taro, N. benthamiana and A. thaliana using RT-PCR and optimum PCR conditions was obtained. Next step was to validate the EF1α and F-box genes as reference gene in virus infected taro using RT-qPCR. The data obtained was analysed statistically to determine any significant variation in the mRNA accumulation of EF1α and F-box between healthy and DsMV infected taro. Both these genes were shown to have similar and constant expression in healthy and DsMV infected taro. This suggests that these genes could be used as suitable reference gene for gene quantification studies in taro. For other species such as N. benthamiana and other monocot and dicots, this would need to be tested and confirmed empirically. For the analysis of PVIP mRNA accumulation in healthy and DsMV infected taro, PVIP primers designed for N. benthamiana were tested on taro and A. thaliana using RT-PCR. A very faint product of expected size was observed for both taro and A. thaliana using this primer pair; however, it was assumed that the observed amplification would be sufficient for the efficient amplification of PVIP in taro for RT-qPCR analysis. The mRNA accumulation of PVIP was normalised against the EF1α and F-box reference genes for the RT-qPCR analysis. However, efficient amplification was not achieved from either healthy or DsMV infected taro using the N. benthamiana specific PVIP primer pair. Hence, generic monocot and dicot PVIP primers need to be designed and tested for future gene quantification studies in taro and other monocot and dicot species, to determine the variation in the mRNA accumulation of PVIP in virus infected plants.