Enhanced whitefly resistance in transgenic tobacco plants expressing double stranded RNA of v-ATPase A gene

Abstract

Background: Expression of double strand RNA (dsRNA) designed against important insect genes in transgenic plants have been shown to give protection against pests through RNA interference (RNAi), thus opening the way for a new generation of insect-resistant crops. We have earlier compared the efficacy of dsRNAs/siRNAs, against a number of target genes, for interference in growth of whitefly (Bemisia tabaci) upon oral feeding. The v-ATPase subunit A (v-ATPaseA) coding gene was identified as a crucial target. We now report the effectiveness of transgenic tobacco plants expressing siRNA to silence v-ATPaseA gene expression for the control of whitefly infestation.

Methodology/principal findings: Transgenic tobacco lines were developed for the expression of long dsRNA precursor to make siRNA and knock down the v-ATPaseA mRNA in whitefly. Molecular analysis and insecticidal properties of the transgenic plants established the formation of siRNA targeting the whitefly v-ATPaseA, in the leaves. The transcript level of v-ATPaseA in whiteflies was reduced up to 62% after feeding on the transgenic plants. Heavy infestation of whiteflies on the control plants caused significant loss of sugar content which led to the drooping of leaves. The transgenic plants did not show drooping effect.

Conclusions/significance: Host plant derived pest resistance was achieved against whiteflies by genetic transformation of tobacco which generated siRNA against the whitefly v-ATPaseA gene. Transgenic tobacco lines expressing dsRNA of v-ATPaseA, delivered sufficient siRNA to whiteflies feeding on them, mounting a significant silencing response, leading to their mortality. The transcript level of the target gene was reduced in whiteflies feeding on transgenic plants. The strategy can be taken up for genetic engineering of plants to control whiteflies in field crops.

Figure 1. Development of transgenic tobacco expressing whitefly v-ATPaseA specific dsRNA.

(A) Physical map of dsRNA expression cassette in pBI101. (B) transgenic and control tobacco plants showing comparable morphology. (C) selection of T₁ seeds on kanamycin (300 mg/l) medium, showing non-transgenic seedlings turning white. (D) PCR analysis of transgenic lines in T₁ generation by amplification of nptII gene (upper panel) and amplification of v-ATPaseA+RTM1 intron (lower panel), M; 100 bp DNA ladder; lanes 2–9, different transgenic tobacco lines, lane 10; positive control.

Figure 2. Expression analysis of v-ATPase A specific RNA in different transgenic lines of tobacco.

Dot-blot assay of total RNA from 12 T₁ transgenic lines with probe of (A) v-ATPaseA gene and (B) U6 gene, number shown are different transgenic lines. (C) Dot-blot assay of RNA from control plants (expressing dsRNA of asal gene); RNA was spotted and hybridised with same probes to show specificity of hybridisation. (D) Northern blot analysis of four selected transgenic lines with v-ATPaseA specific probe. Blot showed positive signal from all tested transgenic lines (Lane 2; line 7, Lane 3; line 11, Lane 4; line 16, Lane 5; line 24) while control line (Lane 1; control) did not show any signal. Loading of equal amounts of RNA was confirmed by ethidium bromide staining.

Figure 3. In-vivo bioassay of transgenic and control plants with whitefly.

Control and transgenic lines challenged with freshly emerged adult whiteflies. (A) Whiteflies colonizing on control plant while the transgenic lines show protection, lower panel shows enlarged view of upper panel (B) The surviving number of insects were counted after 5, 10 and 15 days. The count of whitefly and percent population reduction was plotted for each of the selected lines. Bars = number of insects; lines = population reduction over control. Data shown are average of six plants of each line (3experimental setups ×2 plants/setup) ± standard deviation. Asterisk indicates significant difference in treatments (transgenic plants) compared to control (dsasal) plants (Student's t-test, *p<0.05,**p<0.01).

Figure 4. Drooping of control tobacco plants after whitefly infestation.

The plants were challenged with the insects at age of 3 months (A) and 4.5 months (B). The control plants lost large amount of sap owing to heavy infestation of whitefly and drooped after 15 days, transgenic plant exhibited nearly complete tolerance. Leaves of mature plants started to droop at the base (B, indicated by arrows). (C) The sugar content of the leaves was estimated in the 3-month-old plants. Infestation reduced the sugar content to approximately 50% in the control plants. Asterisk indicates significant difference in sugar content after 15 days of infestation in control lines (Student's t-test, *p<0.05).

Figure 5. Insect bioassay with small RNAs.

Small RNAs (15 µg) was fed to whiteflies after mixing in artificial diet. Graph shows percent mortality of each line (3 biological replicates ×3 technical replicates each) means ± standard deviations. Asterisk indicates significant difference in treatments (transgenic small RNA fractions) compared to control (control small RNA fractions) (Student's t-test, *p<0.05).

Figure 6. Silencing of whitefly v-ATPaseA after feeding on transgenic tobacco by qRT-PCR.

Graph shows significant down-regulation of v-ATPaseA gene of whiteflies after feeding on transgenic lines. Data shown are means ± standard deviations of each line. Asterisk indicates significant difference in treatments compared to control (Student's t-test, *p<0.05, **p<0.01,).