Epigenetic regulation of endogenous plant pararetroviruses

This thesis focuses on epigenetic processes involved in the regulation of gene expression in endogenous pararetroviruses (EPRVs), exemplified by endogenous Petunia vein clearing virus (ePVCV-1) and its episomal form, PVCV. Since ePVCV-1/PVCV was found to have features characteristic of retrotransposon and endogenous retroviruses (Richert-Poggeler and Shepherd, 1997), detailed analysis of these retroelements in different systems gives a deep insight to understand the interconnection of these elements and their regulation by the host cellular machinery as described in chapter one.
Chapter two describes the different silencing states of ePVCV-1 in two distinct Petunia hybrida lines, “white 138” (W138) and “rose du ciel” (Rdc). Despite of ePVCV-1 integration into the pericentromeric regions of the Petunia hybrida chromatin, we found that this position still allows for a low level of transcription that increases with increasing plant age and is higher in W138 than Rdc. To correlate these findings with epigenetic marks, we compared these cultivars in respect to DNA- and histone-methylation and siRNA production. Using bisulfite treatment, ePVCV-1 sequences were found to be methylated at cytosines in all contexts. Astonishingly, however, in both hosts the methylation rate in the non-coding region containing the promoter is relatively low. This might indicate a special ability of the viral promoter to escape complete inactivation by methylation. In Rdc, nearly all histones covering the ePVCV-1 coding region were methylated at lysine 9 of histone 3 (H3K9), a flag for heterochromatin, while in W138 about half of them were of the H3K9- and half of the H3K4-type, the latter representing active chromatin. Interestingly and in accordance with the DNA methylation data, the H3K4/H3K9 ratio was relatively high for the promoter region of both cultivars. The higher H3K4/H3K9 ratio in W138 correlates with an increased rate of ePVCV-1 induction. Furthermore, we show the production of siRNAs of three different size classes (24, 22 and 21 nt) in both cultivars, all of
which are weaker in W138 than in Rdc. Together our observations indicate that W138 is less efficient in silencing of the endogenous viral sequences than Rdc.
In chapter three, I investigated the promoter region of PVCV and determined its ability to direct transcription in transgenic plants. Furthermore, I analyzed the regulatory elements of this particular promoter in comparison with those of other plant pararetrovirus promoters. In particular I studied the functionality of an as-1 like element and its contribution to PVCV promoter expression. Although originally of medium strength, the promoter could be improved to about 50% strength of that of the CaMV 35S promoter by “repairing“ a pair of degenerated as-1 enhancer elements. We show, that the promoter includes upstream and downstream enhancer elements, and that it can be improved considerably by restoring two degenerated as-1 elements.
The concept of creating virus-resistant plants by transformation with genes derived from the pathogen genome is a well-exploited and highly effective procedure to fight viruses as causal agents of diseases in plants (Fichen and Beachy, 1993). Recently it has been demonstrated that RNA interference (RNAi) can be successfully triggered against plant viruses by transient expression of an inverted repeat of target sequences (Pooggin et al., 2003; Tenllado et al., 2004). In chapter four, we use this technique to develop RNA-mediated banana streak virus resistance via TGS and/or PTGS and the method should prevent the outbreak of virus infection upon rare spontaneous induction of endogenous BSV in tissue culture.
Chapter five is a publication in EMBO journal to which I contributed in major ways. This paper describes the production of cloned PVCV originating directly from Petunia plants and from a Petunia gene library. Our findings allowed comparative and direct analysis of horizontally and vertically transmitted virus forms and demonstrated their infectivity using biolistic transformation of a provirus-free petunia species. Some integrants within the genome of P.hybrida were found to be arranged in tandem, allowing direct release of virus by transcription. In addition to known inducers of endogenous pararetroviruses, such as genome hybridization, tissue culture and abiotic stresses, we observed
activation of PVCV after wounding. Our data also support the hypothesis that the host plant uses DNA methylation to control the endogenous pararetrovirus.
In a preamble I point out, which part of this paper is based on my own experimentation and interpretation. on to control the endogenous pararetrovirus.
In a preamble I point out, which part of this paper is based on my own experimentation and interpretation.