Virus-Induced Gene Silencing and Transient Gene Expression in Soybean (Glycine max)
 Using Bean Pod Mottle Virus
 Infectious Clones

Rpp4 (Resistance to Phakopsora pachyrhizi 4) confers resistance to Phakopsora pachyrhizi Sydow, the causal agent of Asian soybean rust (ASR). By combining expression profiling and virus induced gene silencing (VIGS), we are developing a genetic framework for Rpp4-mediated resistance. We measured gene expression in mock-inoculated and P. pachyrhizi-infected leaves of resistant soybean accession PI459025B (Rpp4) and the susceptible cultivar (Williams 82) across a 12-day time course. Unexpectedly, two biphasic responses were identified. In the incompatible reaction, genes induced at 12 h after infection (hai) were not differentially expressed at 24 hai, but were induced at 72 hai. In contrast, genes repressed at 12 hai were not differentially expressed from 24 to 144 hai, but were repressed 216 hai and later. To differentiate between basal and resistance-gene (R-gene) mediated defence responses, we compared gene expression in Rpp4-silenced and empty vector-treated PI459025B plants 14 days after infection (dai) with P. pachyrhizi. This identified genes, including transcription factors, whose differential expression is dependent upon Rpp4. To identify differentially expressed genes conserved across multiple P. pachyrhizi resistance pathways, Rpp4 expression datasets were compared with microarray data previously generated for Rpp2 and Rpp3-mediated defence responses. Fourteen transcription factors common to all resistant and susceptible responses were identified, as well as fourteen transcription factors unique to R-gene-mediated resistance responses. These genes are targets for future P. pachyrhizi resistance research.

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Down-regulation of flavonoid 3′-hydroxylase gene expression by virus-induced gene silencing in soybean reveals the presence of a threshold mRNA level associated with pigmentation in pubescence

Journal of Plant Physiology ◽

10.1016/j.jplph.2008.02.002 ◽

2009 ◽

Vol 166 (1) ◽

pp. 32-39 ◽

Cited By ~ 17

Author(s):

Atsushi Nagamatsu ◽

Chikara Masuta ◽

Hideyuki Matsuura ◽

Keisuke Kitamura ◽

Jun Abe ◽

...

Keyword(s):

Gene Expression ◽

Gene Silencing ◽

Mrna Level ◽

Virus Induced Gene Silencing ◽

Down Regulation ◽

Hydroxylase Gene

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Rapid, high efficiency virus-mediated mutant complementation and gene silencing in Antirrhinum

Plant Methods ◽

10.1186/s13007-020-00683-5 ◽

2020 ◽

Vol 16 (1) ◽

Author(s):

Ying Tan ◽

Alfredas Bukys ◽

Attila Molnár ◽

Andrew Hudson

Keyword(s):

Gene Expression ◽

Gene Silencing ◽

High Efficiency ◽

Tobacco Rattle Virus ◽

Transient Gene Expression ◽

Mutant Phenotype ◽

Homologous Sequence ◽

Homologous Gene ◽

Loss Of Function ◽

H Protein

Abstract Background Antirrhinum (snapdragon) species are models for genetic and evolutionary research but recalcitrant to genetic transformation, limiting use of transgenic methods for functional genomics. Transient gene expression from viral vectors and virus-induced gene silencing (VIGS) offer transformation-free alternatives. Here we investigate the utility of Tobacco rattle virus (TRV) for homologous gene expression in Antirrhinum and VIGS in Antirrhinum and its relative Misopates. Results A. majus proved highly susceptible to systemic TRV infection. TRV carrying part of the Phytoene Desaturase (PDS) gene triggered efficient PDS silencing, visible as tissue bleaching, providing a reporter for the extent and location of VIGS. VIGS was initiated most frequently in young seedlings, persisted into inflorescences and flowers and was not significantly affected by the orientation of the homologous sequence within the TRV genome. Its utility was further demonstrated by reducing expression of two developmental regulators that act either in the protoderm of young leaf primordia or in developing flowers. The effects of co-silencing PDS and the trichome-suppressing Hairy (H) gene from the same TRV genome showed that tissue bleaching provides a useful marker for VIGS of a second target gene acting in a different cell layer. The ability of TRV-encoded H protein to complement the h mutant phenotype was also tested. TRV carrying the native H coding sequence with PDS to report infection failed to complement h mutations and triggered VIGS of H in wild-type plants. However, a sequence with 43% synonymous substitutions encoding H protein, was able to complement the h mutant phenotype when expressed without a PDS VIGS reporter. Conclusions We demonstrate an effective method for VIGS in the model genus Antirrhinum and its relative Misopates that works in vegetative and reproductive tissues. We also show that TRV can be used for complementation of a loss-of-function mutation in Antirrhinum. These methods make rapid tests of gene function possible in these species, which are difficult to transform genetically, and opens up the possibility of using additional cell biological and biochemical techniques that depend on transgene expression.

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