Potential of SiRNA and Artificial MiRNAs Against Cotton Leaf Curl Burewala Virus V2 Gene Yielding Resistance to Begomoviruses

Abstract

Diseases of crop plants caused by begomoviruses (whitefly-transmitted viruses of the family Geminiviridae) are a major constraint to productivity across the warmer parts of the world. Cotton leaf curl disease (CLCuD) across Pakistan and northwestern India has caused severe losses to cotton cultivation since the early 1990s. In Pakistan the disease at this time is caused by a single begomovirus, Cotton leaf curl Burewala virus (CLCuBuV), and a betasatellite, Cotton leaf curl Multan betasatellite (CLCuMuB). Efforts to prevent losses due to CLCuD rely on the use of insecticides to control the vector whitefly and the use of tolerant cotton varieties; no immune varieties so far having been identified. RNAi technology offers a possible mechanism of rapidly developing resistant crop varieties to counter diseases caused by plant-infecting viruses. Here antisense RNA and artificial micro (ami)RNA have been investigated for their potential to yield resistance to CLCuBuV. A major challenge to use of RNAi is the need to identify the best target sequence. Here three fragments of the virion-sense gene V2 of CLCuBuV have been transformed into Nicotiana benthamiana in antisense orientation and assessed for their ability to yield resistance against CLCuBuV and three heterologous begomoviruses. The results are consistent with the idea that RNAi is a homology-based response with transgenic plants showing levels of resistance that correlate with the levels of sequence identity between the transgene and the inoculated virus. However, only for CLCuBuV was resistance at near immunity levels with the V2 sequence closest to the promoter providing the best resistance. Nevertheless, with all three constructs, transgenic plants inoculated with CLCuBuV showed no symptoms, or recovered from initial mild symptoms, and viral DNA levels were low. Additionally, inoculation of CLCuBuV with the CLCuD-associated betasatellite CLCuMuB to transgenic plants did not significantly affect the outcome although it increased the numbers of plants in which viral DNA could be detected, suggesting that the betasatellite may impair RNAi resistance. This effect is likely due to the betasatellite encoding a strong suppressor that inhibits RNAi at both the transcriptional and post - transcriptional levels. xiv Earlier studies have shown that the sequences of naturally occurring miRNA genes can be changed to alter the messenger RNAs that they bind to. Here a cotton microRNA gene (miR169a) was altered to replace the sequence of the mature miRNA with 21 nucleotides of sequence from the V2 gene of CLCuBuV and transformed into N. benthamiana. Two constructs were produced. In one construct (P1CN) the sequence of the miRNA backbone, with the exception of the 21 nucleotides, was left unchanged. In the other (P1DM) the sequence of the backbone was changed to, at least in part, restore the secondary structure of the immature miRNA (referred to as a precursor - miRNA). Inoculation of plants with a range of begomoviruses showed P1CN to give efficient resistance against the homologous virus (CLCuBuV) but not against heterologous viruses. Overall the levels of resistance exhibited depended upon the levels of sequence identity to the target (21nt) sequence, although other factors also likely play a part. For a small number of P1CN plants inoculated with CLCuBuV symptoms were initially evident but the plants recovered and contained low levels of viral DNA. In contrast, transgenic plants inoculated with heterologous viruses showed a greater number of plants symptomatically infected, that did not recover and showed high levels of viral DNA although lower than in infected non-transgenic plants. Transgenic plants harbouring P1DM showed poor resistance to CLCuBuV and little resistance to the heterologous viruses, indicating that the backbone sequence of the premiRNA is important for the biogenesis of mature miRNA. The results indicate that both antisense-RNA and amiRNA have the potential to deliver resistance against begomoviruses. The significance of the results are discussed.