RNA interference-based resistance against a legume mastrevirus.

Area of Science:

• Plant Pathology
• Molecular Biology
• Biotechnology

Background:

• RNA interference (RNAi) is a gene silencing mechanism used for plant virus resistance.
• Its efficacy against DNA viruses, particularly Geminiviridae, is under investigation.
• Dicot-infecting mastreviruses, like CpCDPKV, remain a challenge for RNAi strategies.

Purpose of the Study:

• To investigate the potential of RNA interference (RNAi) for engineering resistance against a dicot-infecting mastrevirus.
• To assess the effectiveness of a hairpin RNAi construct targeting CpCDPKV in Nicotiana benthamiana.
• To evaluate viral DNA accumulation and symptom development in transgenic plants.

Main Methods:

• Development of a hairpin (hp)RNAi construct targeting CpCDPKV genes.
• Stable transformation of Nicotiana benthamiana with the hpRNAi construct.
• Challenge inoculation of transgenic and non-transgenic plants with CpCDPKV.
• Assessment of viral DNA presence using Southern hybridization and real-time quantitative PCR.

Main Results:

• Non-transgenic plants exhibited typical CpCDPKV infection symptoms within two weeks.
• Transgenic plants harboring the hpRNAi construct showed no symptoms of infection.
• Southern hybridization detected no viral DNA in inoculated transgenic plants.
• Real-time quantitative PCR revealed very low viral DNA accumulation in transgenic plants.

Conclusions:

• RNAi-based strategies are effective in protecting plants against dicot-infecting mastreviruses.
• The hpRNAi construct successfully conferred resistance to CpCDPKV in Nicotiana benthamiana.
• Impaired viral replication and/or movement is suggested by the absence of symptoms and low viral loads in transgenic plants.