Artificial microRNA mediated transgenic resistance against Sugarcane mosaic virus (SCMV) in Nicotiana benthamiana

Abstract

Sugarcane (Saccharum spp.) is a major sugar crop in Pakistan, cultivated on about 1.3 million hectares. Mosaic (caused by Sugarcane mosaic virus (SCMV)) is amongst the major diseases of sugarcane in the country, a decline due to mosaic was recorded in cane yield from 10-32% and in sugar yield from 6-10%. Developing transgenic resistance in monocotyledonous crops against pathogens remains a challenging area of research. The objectives of present study were: to determine the virus isolate from local sugarcane varieties; and to analyze transgenic expression of artificial microRNA (amiRNA) and hairpin RNA (hpRNA), targeting simultaneously CP (Coat Protein) and Hc-Pro (Helper Component-Proteinase) genes of SCMV, in Nicotiana tobacum and rice plant. Out of thirty-two symptomatic sugarcane leaf sample, collected from Punjab and Khyberpakhtunkhwa (KPK) provinces, Pakistan during 2013-2014, twenty samples were PCR amplified using CP gene universal primer pairs. These products were cloned and sequenced in both directions. These nucleotide sequences shared highest nucleotide sequence identity between 85-100% nt identity to an isolate of SCMV. Conserved nucleotide sequences, exclusive for DAG and KITC motifs, derived from SCMV CP and Hc-Pro genes were used to develop amiRNA constructs (CP-amiR and Hc-Pro-amiR). These conserved nucleotide sequences were also fused together and assembled into the hpRNA cassette under maize ubiquitin and 35S promoters, to form hpRNA constructs (Ubi-hpCP:Hc-Pro and 35S-hpCP:Hc-Pro). The 35S-GUS:CP:Hc-Pro served as a target reporter gene construct. amiRNA constructs were mobilized into Nicotiana tobacum through Agrobacterium mediated transformation. Only two transgenic lines (CP-amiR-15 and Hc-Pro-amiR 7) showed highest gene expression (about 0.08% and 0.27%, respectively) amongst the other expressing lines. hpRNA constructs delivered into rice callus tissues and N. benthamiana through particle bombardment and Agro-infiltration, respectively, both Ubi-hpCP:Hc-Pro and 35ShpCP:Hc-Pro constructs induced strong silencing of 35S-GUS:CP:Hc-Pro transgene. Transgenic rice plants, containing Ubi-hpCP:Hc-Pro construct, expressed high level of 21-24 nt small interfering RNAs, which induced efficient silencing against the GUS:CP:Hc-Pro transgene delivered by particle bombardment and conferred strong silencing by mechanically inoculated SCMV. It is concluded that fusion hpRNA approach is an effective and affordable method for developing resistance against SCMV in model rice plant and it could confer SCMV resistance when transformed into sugarcane.