e-mail: anhxuan0909@yahoo.com

Advisor: Neena Mitter

RNA silencing has been regarded as a natural self-defense mechanism to protect plants from their pathogen invasion. Thus, there has been a variety of gene silencing strategies developed and exploited the natural pathway to generate virus-resistant plants. The newly developed artificial microRNA technology is generally recognized as an effective method of down-regulating target genes. Nucleocapsid protein (N) and the silencing suppressor (NSs) transcripts, which were important for viral pathogenesis of Tomato spotted wilt virus (TSWV), were targeted with amiR159-based construct in Arabidopsis thaliana ecotype Columbia-O and NicotianaTabacumto combat against such a destructive and pathogenic plant virus.  With the aim of improving the expression of the artificial microRNAs in plants to confer the increased virus resistance, a diversified number of amiRNAs designed were different from the target regions on viral transcripts and the presence or absence of mismatches at nucleotide positions 12, 13 in the complementary strand (amiRNA*).  The expression of these virus-specific amiRNAs in plants was determined by Northern blot analysis. The study results showed that amiTWSV constructs were successfully expressed in the two plant models with the presence of amiRNAs targeting N and NSs transcripts. In addition, the incorporation of the mismatches at nucleotide positions 12, 13 into the complementary strand of amiRNA duplex was not clear to be useful for amiRNAs biogenesis. Notably, the sequence within amiRNA duplex was believed to play a role in its expression and determine the fate of amiRNA and amiRNA* strand. These findings should be beneficial for designing more effective artificial gene silencing agents.

RNA silencing. RNA silencing has been regarded as a natural self-defense mechanism to protect plants from their pathogen invasion. Thus, there has been a variety of gene silencing strategies developed and exploited the natural pathway to generate virus-resistant plants. The newly developed artificial microRNA technology is generally recognized as an effective method of down-regulating target genes. Nucleocapsid protein (N) and the silencing suppressor (NSs) transcripts, which were important for viral pathogenesis of Tomato spotted wilt virus (TSWV), were targeted with amiR159-based construct in Arabidopsis thaliana ecotype Columbia-O and NicotianaTabacumto combat against such a destructive and pathogenic plant virus.  With the aim of improving the expression of the artificial microRNAs in plants to confer the increased virus resistance, a diversified number of amiRNAs designed were different from the target regions on viral transcripts and the presence or absence of mismatches at nucleotide positions 12, 13 in the complementary strand (amiRNA*).  The expression of these virus-specific amiRNAs in plants was determined by Northern blot analysis. The study results showed that amiTWSV constructs were successfully expressed in the two plant models with the presence of amiRNAs targeting N and NSs transcripts. In addition, the incorporation of the mismatches at nucleotide positions 12, 13 into the complementary strand of amiRNA duplex was not clear to be useful for amiRNAs biogenesis. Notably, the sequence within amiRNA duplex was believed to play a role in its expression and determine the fate of amiRNA and amiRNA* strand. These findings should be beneficial for designing more effective artificial gene silencing agents.