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United States Department of Agriculture

Agricultural Research Service

Title: Analysis of Methylation and Post Transcriptional Gene Silencing in Embryos and Seedlings of C5, a Transgenic Plum Pox Virus Resistant Plum

Authors
item Scorza, Ralph
item Webb, Kevin
item Ravelonandro, Michel - INRA, FRANCE

Submitted to: Meeting Proceedings
Publication Type: Proceedings
Publication Acceptance Date: May 15, 2004
Publication Date: May 22, 2004
Citation: Scorza, R., Webb, K.K., Ravelonandro, M. 2004. Analysis of methylation and post transcriptional gene silencing in embryos and seedlings of C5, a transgenic plum pox virus resistant plum. Proceedings of the 2004 International Rosaceae Genome Mapping Conference. p. 20.

Technical Abstract: We have previously shown that in the plum pox virus (PPV)-resistant transgenic plum clone C5 the PPV-coat protein (CP) insert is post-transcriptionally silenced (PTGS) and methylated (Scorza et al., Transgenic Res. 1021-1029, 2001). We have also demonstrated that the transgene insert and PPV resistance are mutually inherited in progeny of C5 (Ravelonandro et al., Acta Hort. 478:67-71, 1998; Scorza et al., Acta Hort. 472:421-427, 1998). In herbaceous systems, PTGS has been shown to be 're-set' following seed germination, generally being reinitiated upon virus infection. To our knowledge, this phenomenon has not been studied in woody perennials. P. domestica is a woody perennial that normally requires stratification (moist chilling) for germination. Seeds of open-pollinated C5 carrying the PPV-CP insert were germinated with and without stratification, as were seeds of C3, a non-PTGS PPV-CP transgenic clone that is susceptible to PPV. Seeds or seedlings used in this study were not inoculated with PPV. The PPV-CP transgene in seeds and germinated seedlings of C3 was not methylated and mRNA production was similar to levels found in leaves from mature C3 trees. No siRNA was detected in C3. DNA analyses showed that at one month following germination the PPV-CP gene in C5 was methylated in seedlings from both stratified and non-stratified treatments at levels comparable to the levels in leaves of the C5 parent, whereas the NPTII and uidA transgenes were unmethylated as expected. C5 embryos and seedlings tested at 2 weeks post-germination showed reduced levels of methylation and increased PPV-CP transgene mRNA expression when compared with 4-week post-germination samples. Short interfering (si) RNA was detected in embryos from ungerminated seeds and from 2-week and 4-week post germination samples as well as from samples of leaves from mature C5 trees. These results indicate that although the expression of PTGS was altered in seeds and in seedlings of C5 two weeks post germination, at one month post germination all components of the PTGS mechanism that were evaluated, including siRNA production, were at levels comparable to those found in leaves of the parental C5 clone. These results demonstrate the transfer of the PTGS mechanism to seedlings of C5 and its early activity even in the absence of PPV inoculation, and suggest the value of C5 as a parent in breeding for resistance to PPV.

Last Modified: 11/28/2014