Molecular cloning and functional characterisation of the tomato E3 ubiquitin ligase SlBAH1 gene.

Emerging evidence suggests that E3 ligases play critical roles in diverse biological processes, including pathogen resistance in plants. In the present study, an ubiquitin ligase gene (SlBAH1) was cloned from a tomato plant, and the functions of the gene were studied. The SlBAH1 gene contained 1002 nucleotides and encodes a protein with 333 amino acids. The SlBAH1 protein contains a SPX domain and a RING domain. SlBAH1 displayed E3 ubiquitin ligase activity in vitro. SlBAH1 was shown to localise in the nucleus, cytoplasm and plasma membrane by a subcellular localisation assay. The expression of SlBAH1 was induced by various hormones and Botrytis cinerea Pers. treatment. SlBAH1-silencing in plants obtained by virus-induced gene silencing (VIGS) technology enhanced resistance to B. cinerea, and the expression of pathogenesis-related (PR) genes, including PR1, PR2, PR4, PR5, and PR7, was significantly increased. These results indicate that the SlBAH1-dependent activation of defence-related genes played a key role in the enhanced fungal resistance observed in the SlBAH1-silenced plants and may be related to the SA-dependent and JA-dependent signalling pathways.

Effects of the sequence characteristics of miRNAs on multi-viral resistance mediated by single amiRNAs in transgenic tobacco.

Artificial microRNA (amiRNA) has become the preferred viral defence that can be induced in plants. In this study, nine amiRNA target sites were selected that were based on the sequence characteristics of natural miRNAs in the cylindrical inclusion protein (CI), nuclear inclusion a protein (NIa), nuclear inclusion b protein (NIb), and coat protein (CP) genes of Potato virus Y (PVY(N)). These amiRNAs that exhibited high similarities to the sequences of PVY(N) and TEV-SD1 were considered. To study the effectiveness of gene silencing in amiRNA-mediated viral resistance, we constructed nine amiRNA plant expression vectors by replacing the functional sequences of miRNA319a precursors with our selected amiRNA sequences. These constructs were subsequently introduced to tobacco plants. A Northern blot assay verified that the nine amiRNA plant expression vectors could successfully express amiRNAs in plants. The analysis of viral resistance demonstrated that these transgenic tobacco plants could effectively inhibit PVY(N) and TEV-SD1 viral infections. The amiRNA that targeted the NIb and CP genes displayed a higher silencing efficiency than did the amiRNAs targeted CI and NIa genes. Northern blot analysis demonstrated that silencing was induced by the original amiRNAs and could be bilaterally extended by the siRNA pathway. That is, the amiRNA and the secondary siRNA mediated the degradation of viral RNA together. Genetic analysis demonstrated that the trait for viral resistance in transgenic plants can be consistently inherited via a single copy of the transgenic sequence. Considering the correlation between the sequence characteristics and the activity of amiRNA, we concluded that a few mismatched bases between the amiRNA and the target sequence could be allowed, particularly the mismatched bases in the 3' end of the amiRNA.

Bacterially expressed double-stranded RNAs against hot-spot sequences of tobacco mosaic virus or potato virus Y genome have different ability to protect tobacco from viral infection.

Posttranscriptional gene silencing, also known as RNA interference, involves degradation of homologous mRNA sequences in organisms. In plants, posttranscriptional gene silencing is part of a defense mechanism against virus infection, and double-stranded RNA is the pivotal factor that induces gene silencing. In this paper, we got seven hairpin RNAs (hpRNAs) constructs against different hot-spot sequences of Tobacco mosaic virus (TMV) or Potato virus Y (PVY) genome. After expression in Escherichia coli HT115, we extracted the seven hpRNAs for the test in tobacco against TMV or PVY infection. The data suggest that different hpRNAs against different hot-spot sequences of TMV or PVY genome had different ability to protect tobacco plants from viral infection. The resistance to TMV conferred by the hpRNA against the TMV movement protein was stronger than other TMV hpRNAs; the resistance to PVY conferred by the hpRNA against the PVY nuclear inclusion b was better than that induced by any other PVY hpRNAs. Northern blotting of siRNA showed that the resistance was indeed an RNA-mediated virus resistance.

[Genetic analysis of RNA-mediated virus resistance in transgenic tobacco].

RNA-mediated virus resistance is an effective way to obtain virus resistant plants and is regarded as a potential strategy with application value in plant resistant virus breeding because of its advantage of high biosafety and long duration. Whether the resistance can inherit stably is a critical factor affecting its application in agriculture. In this paper, several T0 progeny transgenic plants with different resistant levels and with different transgene copy numbers were selected for further study. The results showed that the inheritance of the transgene in T0 susceptible transgenic plants containing 1 -2 transgene copies followed Mendelian segregation pattern of single gene inheritance in their progenies. Resistant transgenic plants, which contained 4 -6 transgene copies, followed 15:1 and 63:1 segregation patterns in the T1 progeny. Transgene recombination and re-organization in the plant genome was observed, which resulted in unstable inheritance of the transgene in the progenies. Homozygous resistant transgenic plants were obtained after 3 to 4 generations in the progeny. Analysis of the transgene integration pattern indicated that most of the highly resistant plants contained inverted repeat (IR) sequences of the transgene.