Silencing of <i>HaAce1</i> gene by host-delivered artificial microRNA disrupts growth and development of <i>Helicoverpa armigera</i>

<div><p>The polyphagous insect-pest, <i>Helicoverpa armigera</i>, is a serious threat to a number of economically important crops. Chemical application and/or cultivation of <i>Bt</i> transgenic crops are the two strategies available now for insect-pest management. However, environmental pollution and long-term sustainability are major concerns against these two options. RNAi is now considered as a promising technology to complement <i>Bt</i> to tackle insect-pests menace. In this study, we report host-delivered silencing of <i>HaAce1</i> gene, encoding the predominant isoform of <i>H</i>. <i>armigera</i> acetylcholinesterase, by an artificial microRNA, <i>HaAce1</i>-amiR1. Arabidopsis pre-miRNA164b was modified by replacing miR164b/miR164b* sequences with <i>HaAce1</i>-amiR1/<i>HaAce1</i>-amiR1* sequences. The recombinant <i>HaAce1</i>-preamiRNA1 was put under the control of CaMV 35S promoter and NOS terminator of plant binary vector pBI121, and the resultant vector cassette was used for tobacco transformation. Two transgenic tobacco lines expressing <i>HaAce1</i>-amiR1 was used for detached leaf insect feeding bioassays. Larval mortality of 25% and adult deformity of 20% were observed in transgenic treated insect group over that control tobacco treated insect group. The reduction in the steady-state level of <i>HaAce1</i> mRNA was 70–80% in the defective adults compared to control. Our results demonstrate promise for host-delivered amiRNA-mediated silencing of <i>HaAce1</i> gene for <i>H</i>. <i>armigera</i> management.</p></div

Expression of <i>HaAce1</i>-amiR1 in transgenic tobacco lines and down regulation of <i>HaAce1</i> in <i>H</i>. <i>armigera</i> adults.

<p>(A) Northern blot showing <i>HaAce1</i>-amiR1 expression in the transgenic tobacco lines, 17R1 and 29R1. Small RNAs were hybridized with DIG end labelled Anti-amiR<i>Ace</i> probe of 21 nt. C, pBI121 transformed tobacco (vector control); 29R1 & 17R1, <i>HaAce1</i>-amiR1 expression in the selected two transgenic tobacco line, 29R1 and 17R1, respectively; P, labelled Anti-amiR<i>Ace</i> probe. (B) Relative expression of <i>HaAce1</i> in the <i>H</i>. <i>armigera</i> adults. Vector control, <i>HaAce1</i> transcript abundance in <i>H</i>. <i>armigera</i> adults emerged from vector control tobacco treated group; 17R1 and 29R1, <i>HaAce1</i> transcript abundance in deformed <i>H</i>. <i>armigera</i> adults emerged from 17R1 and 29R1 transgenic tobacco treatment groups, respectively. <i>β</i>-<i>Actin</i> gene of <i>H</i>. <i>armigera</i> was used as an internal control. Real time PCR data was analysed using delta-delta Ct method. One way ANOVA test was used to perform the statistical analysis of the data. *** Extremely significant at P<0.001. The test was performed three times.</p

Designing and construction of pBI::HAR1.

<p>Primer extension PCR was done with a pair of overlapping primers, <i>HAR-</i>F & <i>HAR</i>-R (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194150#pone.0194150.s006" target="_blank">S1 Table</a>) containing <i>Xba</i>I and <i>Sac</i>I recognition sequences at their 5′ ends. <i>Xba</i>I and <i>Sac</i>I digested PCR product was ligated onto pUC19. pBI121 backbone was generated by digestion with <i>Xba</i>I and <i>Sac</i>I that released <i>GUS</i> coding sequence. After sequence confirmation, <i>HaAce1</i>-preamiRNA1 DNA fragment was released from recombinant pUC19 and ligated onto pBI121 backbone to get pBI::HAR1.</p

Mortality and developmental deformity in <i>H</i>. <i>armigera</i>.

<p>Thirty second instar larvae of <i>H</i>. <i>armigera</i> were fed continuously on each transgenic line (Line 17R1 and 29R1) and vector control separately (one larva per plate) till their active feeding stage<b>.</b> (A) Mortality percentage of <i>H</i>. <i>armigera</i> larvae. Vector control, mortality percentage in larvae fed on pBI121 transformed tobacco leaves; 17R1, mortality percentage in larvae fed on 17R1 transgenic tobacco line; 29R1, mortality percentage in larvae fed on 29R1 transgenic tobacco line. (B) Deformity percentage of <i>H</i>. <i>armigera</i> adults. Vector control, percentage of emergence of deformed adults from larvae fed on pBI121 transformed tobacco leaves; 17R1, percentage of emergence of deformed adults from larvae fed on 17R1 transgenic tobacco line; 29R1, percentage of emergence of deformed adults from larvae fed on 29R1 transgenic tobacco line. (C) Phenotype of <i>H</i>. <i>armigera</i> adults. Upper panel: Normal adults developed from vector control fed larvae; Lower panel: Deformed adults developed from transgenic fed larvae. One way ANOVA test was used to perform statistical analysis of the data. *** denotes extremely significant differences at P<0.001. The test was repeated three times.</p