Long term efficacy and safety of RNAi-mediated virus resistance in 'Honeysweet' plum

Authors: SINGH, KHUSHWANT - Crop Research Institute - Czech Republic; Callahan, Ann; SMITH, BRENDA - Oklahoma State University; MALINOWSKI, TADEUSZ - Research Institute Of Horticulture; SCORZA, RALPH - Retired ARS Employee; JAROSOVA, JANA - Crop Research Institute - Czech Republic; BEONI, EVA - Crop Research Institute - Czech Republic; POLAK, JAROSLAV - Crop Research Institute - Czech Republic; KUNDU, JIBAN KUMAR - Crop Research Institute - Czech Republic; Dardick, Christopher - Chris

Submitted to: Frontiers in Plant Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/7/2021
Publication Date: 10/12/2021
Citation: Singh, K., Callahan, A.M., Smith, B., Malinowski, T., Scorza, R., Jarosova, J., Beoni, E., Polak, J., Kundu, J., Dardick, C.D. 2021. Long term efficacy and safety of RNAi-mediated virus resistance in 'Honeysweet' plum. Frontiers in Plant Science. 12:726881. https://doi.org/10.3389/fpls.2021.726881.
DOI: https://doi.org/10.3389/fpls.2021.726881

Interpretive Summary: ‘HoneySweet’ plum is resistant to plum pox virus because a gene for the virus was inserted into the plum DNA. This gene triggered a natural immune response to viruses known as RNAi such that invading virus is recognized and destroyed. The trees have been grown in European field trials as well as the US for over 20 years. This study examined the effects the long-term growth had on the resistance as well as the safety of the transgenic tree and resulting fruit. Despite continual infection pressure through an infected graft, ‘HoneySweet’ trees remained virus free throughout the monitoring period from 2002-2017. The presence of the virus gene in ‘HoneySweet’ did not cause the virus in the graft to change or become more infective. The new RNAs produced by ‘HoneySweet’ were at least 10=100 times less than similar RNAs produced by a plum pox virus infected tree which can be sold in the market. Feeding studies with mice had no negative effects on the health or size of the mice. Overall, these studies supported the safety of this approach to virus resistant plants for both plant health and human health.

Technical Abstract: Interfering RNA (RNAi) technology has been established as an effective strategy to protect plants against viral infection. Despite this success, RNAi has rarely been applied due to the regulatory barriers that confront genetically engineered plants and growing concerns over potential environmental and health risks posed by non-endogenous small RNAs. ‘HoneySweet’ was developed as a virus resistant plum variety that is protected by an RNAi-mediated process against Sharka disease caused by plum pox virus infection. ‘Honeysweet’ has been approved for cultivation in the United States but not in countries where plum pox virus is endemic. Here we evaluated the long-term efficacy of virus resistance in ‘Honeysweet’, the nature and stability of its sRNA profile, and the potential health risks of consuming ‘HoneySweet’. Graft infected ‘Honeysweet’ trees carrying large non-transgenic infected limbs remained virus free after >10 years in the field and viral sequences from the non-transgenic infected limbs showed no evidence of adaptation to the RNAi-based resistance. Small RNA profiling revealed that transgene-derived sRNA levels were stable across different environments and on average were >10 times lower than those present in symptom-less fruit from virus infected trees. Comprehensive 90-day mouse feeding studies showed no adverse health impacts in mice and there was no evidence for potential siRNA off-target pathologies predicted by comparisons of the most abundant transgene derived sRNAs to the mouse genome. Collectively, the data confirm that RNAi provides a highly effective, stable, and safe strategy to combat virus diseases in crop plants.