Chinese Wheat Mosaic Virus-Induced Gene Silencing in Monocots and Dicots at Low Temperature

Abstract

Virus-induced gene silencing (VIGS) is an important tool for functional genomics studies in plants. With this method, it is possible to target most endogenous genes and downregulate the messenger RNA (mRNA) in a sequence-specific manner. Chinese wheat mosaic virus (CWMV) has a bipartite, single-strand positive RNA genome, and can infect both wheat and Nicotiana benthamiana, and the optimal temperature for systemic infection in plants is 17°C. To assess the potential of the virus as a vector for gene silencing at low temperature, a fragment of the N. benthamiana or wheat phytoene desaturase (PDS) gene was expressed from a modified CWMV RNA2 clone and the resulting photo bleaching in infected plants was used as a reporter for silencing. Downregulation of PDS mRNA was also measured by quantitative reverse-transcriptase polymerase chain reaction (RT-qPCR). In experiments using fragments of PDS ranging from 500 to 1500 nucleotides, insert length influenced the stability and the efficiency of VIGS. The CWMV induced silencing system was also used to suppress miR165/166 and miR3134a through expression of miRNA target mimics. The relative expression levels of mature miR165/166 and miR3134a decreased whereas the transcript levels of their target genes increased. Interestingly, we also found the CWMV-induced silencing system was more efficient compare with the vector based on Barley stripe mosaic virus (BSMV) or Foxtail mosaic virus (FoMV) in wheat or the vector based on TRV in N. benthamiana at 17°C. In summary, the CWMV vector is effective in silencing endogenous genes and miRNAs at 17°C, thereby providing a powerful tool for gene function analysis in both N. benthamiana and wheat at low temperature.

Genome-wide identification of the histone acetyltransferase gene family in Triticum aestivum

“…For example, the most suitable temperature for wheat streak mosaic virus (WSMV) infection is 15 °C [ 54 ], the most suitable temperature for CWMV infection is 17 °C [ 55 ]. Virus accumulation increases at low temperatures [ 56 ]. It is also interesting that WYMV and CWMV have hidden symptoms that leaves turn green at high temperatures of about 24 °C [ 57 ].…”

“…BSMV-based gene vectors were kindly provided by Dr. Dawei Li, China [ 65 ]. CWMV-based and WYMV-based gene vectors were kindly provided by Dr. Jian Yang, China [ 56 , 66 ]. The three viruses have the same inoculation method, and we will take plants inoculated with BSMV as an example.…”

NbWRKY40 Positively Regulates the Response of Nicotiana benthamiana to Tomato Mosaic Virus via Salicylic Acid Signaling

“…Approximately 300 bp partial fragments of the NbWRKY40 coding sequence were RT–PCR–amplified using total RNA from N. benthamiana as a template using specific primers ( Supplementary Table 1 ), double–digested using Bam HI and Sma I, and then inserted into the TRV–RNA2 expression vector. Both the resulting TRV: NbWRKY40 and TRV–RNA1 vectors were electroporated into A. tumefaciens GV3101 to knock down NbWRKY40 expression as described previously ( Yang et al, 2018 ). The empty TRV–RNA2 and TRV–RNA1 (TRV:00) were used to generate a negative control.…”

Apple latent spherical virus (ALSV)-induced gene silencing in a medicinal plant, Lithospermum erythrorhizon

“…To determine if ALSV can infect L. erythrorhizon , we assessed its ability to knock down expression of the phytoene desaturase ( PDS ) gene in this plant species. Successful infection and PDS gene silencing should result in the appearance of albino leaves, a representative phenotype of the knockdown of PDS function 15 , 16 .…”