Resistance to Wheat streak mosaic virus generated by expression of an artificial polycistronic microRNA in wheat

Abstract

SummaryWheat streak mosaic virus (WSMV) is a persistent threat to wheat production, necessitating novel approaches for protection. We developed an artificial miRNA strategy against WSMV, incorporating five amiRNAs within one polycistronic amiRNA precursor. Using miRNA sequence and folding rules, we chose five amiRNAs targeting conserved regions of WSMV but avoiding off-targets in wheat. These replaced the natural miRNA in each of five arms of the polycistronic rice miR395, producing amiRNA precursor, FanGuard (FGmiR395), which was transformed into wheat behind a constitutive promoter. Splinted ligation detected all five amiRNAs being processed in transgenic leaves. Resistance was assessed over two generations. Three types of response were observed in T 1 plants of different transgenic families: completely immune; initially resistant with resistance breaking down over time; and initially susceptible followed by plant recovery. Deep sequencing of small RNAs from inoculated leaves allowed the virus sequence to be assembled from an immune transgenic, susceptible transgenic, and susceptible non-transgenic plant; the amiRNA targets were fully conserved in all three isolates, indicating virus replication on some transgenics was not a result of mutational escape by the virus. For resistant families, the resistance segregated with the transgene. Analysis in the T 2 generation confirmed the inheritance of immunity and gave further insights into the other phenotypes. Stable resistant lines developed no symptoms and no virus by ELISA; this resistance was classified as immunity when extracts failed to transmit from inoculated leaves to test plants. This study demonstrates the utility of a polycistronic amiRNA strategy in wheat against WSMV.

Efficient use of artificial micro-RNA to downregulate the expression of genes at the post-transcriptional level in Arabidopsis thaliana

“…Another fundamental aspect of artificial miRNA is that it has been used to downregulate the expression of exogenous genes e.g., the invading virus pathogen in wheat, such as the wheat streak mosaic virus (Fahim et al, 2012).…”

Small Non-Coding RNAs in Plant Immunity

“…Indeed several studies demonstrated that artificial miRNAs (amiRNAs) targeting key components of the viral replication machinery can efficiently impair viral growth upon infection [59][60][61][62][63][64][65]. These efforts have revealed that such resistance is cell-autonomous, inheritable, more efficient than siRNA-mediated strategies and successful in blocking viral replication and movement [59,66].…”

“…These efforts have revealed that such resistance is cell-autonomous, inheritable, more efficient than siRNA-mediated strategies and successful in blocking viral replication and movement [59,66]. Furthermore transgenic plants expressing dimeric or polycistronic amiRNAs directed against different viruses result in a wider spectrum of viral resistance [60,61,63].…”

Recent Biotechnological Advances in the Improvement of Cassava

“…A major limitation of the amiRNA strategy is that the small size of the amiRNA (21nt) increases opportunities for loss of complementarity between the amiRNA and the target gene, and genes from the same family with variations may not be silenced using a single amiRNA. To reduce these risks, a multimeric amiRNA approach in which multiple amiRNAs targeting different conserved regions of the gene can be adopted as has been reported in plant virus control [69,70].…”