Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress

Verona, Italy - 24/27 September, 2003

ISBN 88-900622-4-X

Oral Communication Abstract - S1d

Molecular dissection of TYLCSV Rep-derived resistance mechanisms Illustrates the complexities of VIRUS-HOST interactions

A. Lucioli*, E. Noris**, A. Brunetti*, R. Tavazza*, V. Ruzza*, A. Castillo***, E. Bejarano***, A. Fammartino**, G.P. Accotto**, M. Tavazza*

*) ENEA CR Casaccia, Biotec, Rome, Italy

**) IVV, CNR, Torino, Italy

***) Universidad de Malaga, Spain

dominant negative mutants, silencing, PTGS

Transgenic expression of pathogen-derived sequences has been extensively used to obtain virus-resistant plants. However, not always the predicted molecular interference mechanisms were those actually operating in resistant transgenic plants.

The replication-associated protein (Rep) of geminiviruses is the only viral protein that is absolutely required for viral replication. Rep is involved in several biological processes brought about owing to the presence of distinct functional domains and different states of protein aggregation. We previously exploited the multifunctional character of Rep to develop an interference strategy to impair viral infection. In this respect, we showed that transgenic expression of N-terminal 210 amino acids of Tomato yellow leaf curl Sardinia virus Rep (Rep-210) confers resistance to the homologous virus by inhibiting viral transcription and replication (Brunetti et al. 2001 Journal of Virology).

In the present study, using both biochemical and transgenic approaches, we carry out an extensive investigation of the molecular resistance mechanisms operating in Rep-210 transgenic plants. We show that Rep transcriptional repression does not require the oligomerization domain and that Rep-210 confers resistance through two distinct molecular mechanisms, depending on the challenging virus. Resistance to the homologous virus is achieved by a tight inhibition of viral Rep gene transcription, while that to heterologous virus (Tomato yellow leaf curl virus, Portugal isolate) is due to the interacting property of the Rep-210 oligomerization domain. Moreover, we show that in Rep-210 expressing plants the duration of resistance is related to the unexpected ability of the challenging virus to shut off transgene expression by a post-transcriptional homology-dependent gene silencing mechanism and to spread in virus-silenced plants. According to the ability of TYLCSV to spread in virus-silenced transgenic plants, we show that TYLCSV is able to infect transgenic plants accumulating, before the inoculation, PTGS-generated Rep-210 transgene-specific 21-25 nt small interfering RNAs.

We propose a model in which the resistant or susceptible phenotype can be regarded as the result of a battle where the transgenic plant uses the Rep-210 to repress viral transcription and to assemble dysfunctional oligomers, while the virus induces silencing of the transgene. The impact of TYLCSV-mediated transgene silencing on the development of geminivirus-derived resistances will be also discussed.