Abstract
BackgroundAlthough the draft genome of sorghum is available, the understanding of gene function is limited due to the lack of extensive mutant resources. Virus-induced gene silencing (VIGS) is an alternative to mutant resources to study gene function. This study reports an improved and efficient method for Brome mosaic virus (BMV)-based VIGS in sorghum.MethodsSorghum plants were rub-inoculated with sap prepared by grinding 2 g of infected Nicotiana benthamiana leaf in 1 ml 10 mM potassium phosphate buffer (pH 6.8) and 100 mg of carborundum abrasive. The sap was rubbed on two to three top leaves of sorghum. Inoculated plants were covered with a dome to maintain high humidity and kept in the dark for two days at 18 °C. Inoculated plants were then transferred to 18 °C growth chamber with 12 h/12 h light/dark cycle.ResultsThis study shows that BMV infection rate can be significantly increased in sorghum by incubating plants at 18 °C. A substantial variation in BMV infection rate in sorghum genotypes/varieties was observed and BTx623 was the most susceptible. Ubiquitin (Ubiq) silencing is a better visual marker for VIGS in sorghum compared to other markers such as Magnesium Chelatase subunit H (ChlH) and Phytoene desaturase (PDS). The use of antisense strand of a gene in BMV was found to significantly increase the efficiency and extent of VIGS in sorghum. In situ hybridization experiments showed that the non-uniform silencing in sorghum is due to the uneven spread of the virus. This study further demonstrates that genes could also be silenced in the inflorescence of sorghum.ConclusionIn general, sorghum plants are difficult to infect with BMV and therefore recalcitrant to VIGS studies. However, by using BMV as a vector, a BMV susceptible sorghum variety, 18 °C for incubating plants, and antisense strand of the target gene fragment, efficient VIGS can still be achieved in sorghum.
Highlights
The draft genome of sorghum is available, the understanding of gene function is limited due to the lack of extensive mutant resources
At least two studies report the use of Brome mosaic virus (BMV)-based Virus-induced gene silencing (VIGS) to silence genes in sorghum [25, 26]
The virus was multiplied in N. benthamiana as done in maize VIGS studies with this vector [33] and unlike in Martin et al, [26] and Biruma et al, [25], where BMV viral mRNA was synthesized in vitro to directly infect sorghum plants
Summary
The draft genome of sorghum is available, the understanding of gene function is limited due to the lack of extensive mutant resources. Virus-induced gene silencing (VIGS) is an alternative to mutant resources to study gene function. This study reports an improved and efficient method for Brome mosaic virus (BMV)-based VIGS in sorghum. A current challenge to plant scientists is to increase plant productivity in the changing environment with limited resources such as water and fertile land. Sorghum is a C4 annual crop in the grass family and is characterized by its high photosynthetic efficiency. Sorghum is the fifth most important cereal crop after rice, wheat, maize, and barley [2], with an annual production of about 65.5 million tons from the planted area of 45 million ha worldwide [3].
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