Comparative Analysis of RNAi-Based Methods to Down-Regulate Expression of Two Genes Expressed at Different Levels in Myzus persicae

Abstract

With the increasing availability of aphid genomic data, it is necessary to develop robust functional validation methods to evaluate the role of specific aphid genes. This work represents the first study in which five different techniques, all based on RNA interference and on oral acquisition of double-stranded RNA (dsRNA), were developed to silence two genes, ALY and Eph, potentially involved in polerovirus transmission by aphids. Efficient silencing of only Eph transcripts, which are less abundant than those of ALY, could be achieved by feeding aphids on transgenic Arabidopsis thaliana expressing an RNA hairpin targeting Eph, on Nicotiana benthamiana infected with a Tobacco rattle virus (TRV)-Eph recombinant virus, or on in vitro-synthesized Eph-targeting dsRNA. These experiments showed that the silencing efficiency may differ greatly between genes and that aphid gut cells seem to be preferentially affected by the silencing mechanism after oral acquisition of dsRNA. In addition, the use of plants infected with recombinant TRV proved to be a promising technique to silence aphid genes as it does not require plant transformation. This work highlights the need to pursue development of innovative strategies to reproducibly achieve reduction of expression of aphid genes.

Transmission of Turnip yellows virus by Myzus persicae Is Reduced by Feeding Aphids on Double-Stranded RNA Targeting the Ephrin Receptor Protein

“…Arabidopsis thaliana expressing a hairpin RNA targeting Eph or LacZ as a control (Ara:Hp-Eph and Ara:Hp-LacZ) were described in Mulot et al ( 2016 ) and were grown in an environment-controlled chamber at 23°C day and 20°C night with a 10 h photoperiod as well as Col-0 non-transformed plants. In vitro -synthesized dsRNA targeting Eph or LacZ (dsRNA Eph and dsRNA LacZ ) were obtained as described previously in Mulot et al ( 2016 ).…”

“…M. persicae (Sulzer) colonies were reared on pepper ( Capsicum annuum ) at 20°C with a 16 h photoperiod. Aphids were fed on transgenic A. thaliana or artificially on in vitro -synthesized dsRNA as described in Mulot et al ( 2016 ) except that the acquisition time on the artificial medium containing the dsRNA was extended to 5 days in some experiments and the final dsRNA concentration in the feeding medium was set up to 400 ng/μl in all experiments. When a 5-day acquisition period was performed, the dsRNA-containing medium was replaced after 3 days by a fresh medium containing the dsRNA.…”

“…Total RNA was extracted from whole M. persicae (20 aphids per sample) as described in Mulot et al ( 2016 ). Total RNA was also extracted from 100 dissected guts using the RNeasy Plant Mini Kit (QIAGEN) as described in Mulot et al ( 2016 ) or alternatively from 35 dissected guts using NucleoSpin® RNA XS (Macherey-Nagel). To evaluate Eph -mRNA accumulation, qRT-PCR was performed as in Mulot et al ( 2016 ).…”

“…Total RNA was also extracted from 100 dissected guts using the RNeasy Plant Mini Kit (QIAGEN) as described in Mulot et al ( 2016 ) or alternatively from 35 dissected guts using NucleoSpin® RNA XS (Macherey-Nagel). To evaluate Eph -mRNA accumulation, qRT-PCR was performed as in Mulot et al ( 2016 ). As mentioned in Mulot et al ( 2016 ), the relative expression levels were normalized to Rpl7 and L27 .…”

“…To evaluate Eph -mRNA accumulation, qRT-PCR was performed as in Mulot et al ( 2016 ). As mentioned in Mulot et al ( 2016 ), the relative expression levels were normalized to Rpl7 and L27 . To determine the copy number of TuYV genomes internalized into M. persicae , total RNA was extracted from whole aphids after transferring them for 3 days on non-infected Col-0 (inoculation access period or IAP) to clear the gut lumen.…”