A rapid virus- induced gene silencing (VIGS) method for assessing resistance and susceptibility to cassava mosaic disease

Background: Cassava mosaic disease (CMD) is a major constraint to cassava production in sub- Saharan Africa. Under field conditions, evaluation for resistance to CMD takes 12- 18 months, often conducted across multiple years and locations under pressure from whitefly-mediated transmission. Under greenhouse or laboratory settings, evaluation for resistance or susceptibility to CMD involves transmission of the causal viruses from an infected source to healthy plants through grafting, or by using Agrobacterium- mediated or biolistic delivery of infectious clones. Following inoculation, visual assessment for CMD symptom development and recovery requires 12- 22 weeks. Here we report a rapid screening system for determining resistance and susceptibility to CMD based on virus- induced gene silencing (VIGS) of an endogenous cassava gene. Results: A VIGS vector was developed based on an infectious clone of the virulent strain of East African cassava mosaic virus (EACMV- K201). A sequence from the cassava (Manihot esculenta) ortholog of Arabidopsis SPINDLY (SPY) was cloned into the CP position of the DNA- A genomic component and used to inoculate cassava plants by Helios r Gene Gun microparticle bombardment. Silencing of Manihot esculenta SPY (MeSPY) using MeSPY1- VIGS resulted in shoot- tip necrosis followed by death of the whole plant in CMD susceptible cassava plants within 2- 4 weeks. CMD resistant cultivars were not affected and remained healthy after challenge with MeSPY1- VIGS. Significantly higher virus titers were detected in CMD- susceptible cassava lines compared to resistant controls and were correlated with a concomitant reduction in MeSPY expression in susceptible plants. Conclusions: A rapid VIGS- based screening system was developed for assessing resistance and susceptibility to CMD. The method is space and resource efficient, reducing the time required to perform CMD screening to as little as 2- 4 weeks. It can be employed as a high throughput rapid screening system to assess new cassava cultivars and for screening transgenic, gene edited and breeding lines under controlled growth conditions.