Use of recombinant tobacco mosaic virus to achieve RNA interference in plants against the citrus mealybug, Planococcus citri (Hemiptera: Pseudococcidae)

Abstract

The citrus mealybug, Planococcus citri, is an important plant pest with a very broad plant host range. P. citri is a phloem feeder and loss of plant vigor and stunting are characteristic symptoms induced on a range of host plants, but P. citri also reduces fruit quality and causes fruit drop leading to significant yield reductions. Better strategies for managing this pest are greatly needed. RNA interference (RNAi) is an emerging tool for functional genomics studies and is being investigated as a practical tool for highly targeted insect control. Here we investigated whether RNAi effects can be induced in P. citri and whether candidate mRNAs could be identified as possible targets for RNAi-based P. citri control. RNAi effects were induced in P. citri, as demonstrated by specific target reductions of P. citri actin, chitin synthase 1 and V-ATPase mRNAs after injection of the corresponding specific double-stranded RNA inducers. We also used recombinant Tobacco mosaic virus (TMV) to express these RNAi effectors in Nicotiana benthamiana plants. We found that P. citri showed lower fecundity and pronounced death of crawlers after feeding on recombinant TMV-infected plants. Taken together, our data show that actin, chitin synthase 1 and V-ATPase mRNAs are potential targets for RNAi against P. citri, and that recombinant TMV is an effective tool for evaluating candidate RNAi effectors in plants.

Figure 1. Relative mRNA levels of CHS1 (A) and V-ATPase (B) following microinjection of specific dsRNAs into third instar P. citri.

qRT-PCR results of CHS1 (A) and V-ATPase (B) mRNAs from P. citri following microinjection of elution buffer (EB), green fluorescent protein (GFP), CHS1 or V-ATPase dsRNAs. Total RNA was extracted 4 days post injection and used for cDNA synthesis. qRT-PCR was performed using primers described in Table 3. The P. citri 18S ribosomal RNA was used as an endogenous control in all experiments. Calculation of mRNA levels between control and treated groups was done by the comparative CT or 2^–ΔΔCt method and analyzed by ANOVA using statistix 8.1 software. Numbers followed by the same letter are not different at p<0.01 (LSD 0.08). All experiments were performed twice and data were used collectively.

Figure 2. Detection of P. citri CHS1 and V-ATPase mRNAs (A) and accumulation of RNAi-induced siRNAs (B) in N. benthamiana plants after TMV-CHS1 inoculation.

Plants were inoculated with recombinant TMV containing P. citri CHS1 (TMV-CHS1-S) and V-ATPase (TMV-V-ATPase-S). At 7 days post inoculation, total and small RNAs were isolated from infected plants and analyzed by RT-PCR and siRNA northern blot hybridization, respectively. A) One step RT-PCR was performed using total RNA as a template and CHS1 and V-ATPase primers and products analyzed on the gel. Lane L: 1Kb plus DNA ladder, Lane 1: CHS1 primers and RNA of TMV-CHS1-S inoculated N. benthamiana plants; Lane 2: CHS1 primers and RNA of TMV inoculated plant; Lane 3: V-ATPase primers and RNA of TMV-V-ATPase-S inoculated plant and Lane 4: V-ATPase primers and RNA of TMV inoculated plant. B) Small RNAs were separated by PAGE in 15% acrylamide, 8 M urea gels and processed for northern blot hybridization using ³²P-UTP-labeled negative strand P.citri CHS1 transcript as a probe. Lane 1: TMV-CHS1-S inoculated plant; Lane 2: TMV-CHS1-AS iinoculated plant; Lane 3: TMV-inoculated plant.

Figure 3. Relative levels of CHS1 (A) and V-ATPase (B) mRNAs in P. citri after feeding on N. benthamiana plants inoculated with recombinant TMV expressing P. citri CHS1 and V-ATPase fragments.

qRT-PCR results of CHS1 (A) and V-ATPase (B) mRNA levels from P. citri after feeding on N. benthamiana plants infected with recombinant TMVs. TMV-GFP (pJL24) was used as a control and sense and antisense inserts of P. citri CHS1/V-ATPase sequences were compared. Total RNA was extracted after 12 days feeding on plants and used for cDNA synthesis. qRT-PCR was performed using primers described in Table 3. The 18S ribosomal RNA was used as an endogenous control in all experiments. Calculation of mRNA levels between control and treated groups was carried out by the comparative CT or 2^–ΔΔCt method and analyzed by ANOVA using statistix 8.1 software. Numbers with the same letter indicate homogenous groups at p<0.01 (LSD 0.04 and 0.21) for CHS1 and V-ATPase respectively. All experiments were performed twice and data were used collectively.

Figure 4. P. citri feeding on N. benthamiana plants inoculated with TMV-Actin.

Mealybug crawlers feeding on: A) N. benthamiana plants inoculated with TMV only (pJL36) showing healthy crawlers emerging while B, C and D show N. benthamiana plants inoculated with recombinant TMV (TMV-Actin) and show a very high mortality in crawlers and adults. Arrows in A indicate healthy crawlers; in B & C arrows indicate dead crawlers and D the arrows indicate dead adults.

Figure 5. Ovisac production and survival of adult P. citri fed on plants inoculated with different recombinant TMV constructs.

Number of surviving adult P. citri and ovisac production following 12 days feeding on plants inoculated with different recombinant TMV constructs. HC  =  healthy control N. benthamiana plants; TMV-GFP  =  TMV carrying GFP (TMV JL24); TMV  =  TMV with no insert (pJL36); TMV-Actin  =  part of the actin RNA inserted into TMV; TMV-CHS1-S and TMV-CHS1-AS  =  the chitin synthase 1 (CHS1) RNA fragment inserted into TMV in sense and antisense orientations, respectively; TMV-V-ATPase-S and TMV-V-ATPase-AS  =  the V-ATPase RNA fragment inserted into TMV in sense and antisense orientations, respectively. Numbers indicated with the same letter are homogenous groups at p<0.05.

Figure 6. Emergence of P. citri crawlers and their survival on plants inoculated with different recombinant TMV constructs.

Comparison of alive and total crawlers that emerged 18P. citri onto plants inoculated with the following viral constructs: HC  =  healthy control N. benthamiana plants; TMV-GFP  =  TMV carrying GFP (TMV JL24); TMV  =  TMV with no insert (pJL36); TMV-Actin  =  part of the actin RNA inserted into TMV; TMV-CHS1-S and TMV-CHS1-AS  =  the chitin synthase 1 (CHS1) RNA fragment inserted into TMV in sense and antisense orientations, respectively; TMV-V-ATPase-S and TMV-V-ATPase-AS  =  the V-ATPase RNA fragment inserted into TMV in sense and antisense orientations, respectively. Numbers indicated with the same letter are homogenous groups at p<0.05.

Figure 7. P. citri feeding on N. benthamiana plants inoculated with TMV-CHS1 and TMV-V-ATPase.

Mealybug crawlers feeding on: A) N. benthamiana plants infected with TMV (pJL36) show healthy crawlers emerging while B and C show N. benthamiana plants infected with TMV-CHS1-S where B shows abnormal ovisac formation and C shows a high mortality of crawlers. D shows an N. benthamiana plant infected with TMV-V-ATPase-S showing a high mortality in crawlers. Arrows in A indicate healthy crawlers, in B an abnormal ovisac and in C & D indicate dead crawlers.

Figure 8. Mortality in P. citri adults and crawlers on plants inoculated with different recombinant TMV constructs.

Comparison of corrected mortality in adult P. citri and crawlers following 18 days after release of insects onto plants inoculated with the following viral constructs: HC: healthy control N. benthamiana plant; TMV-GFP  =  TMV carrying GFP (TMV JL24); TMV  =  TMV with no insert (pJL36); TMV-Actin  =  part of the actin RNA inserted into TMV; TMV-CHS1-S and TMV-CHS1-AS  =  the chitin synthase 1 (CHS1) RNA fragment inserted into TMV in sense and antisense orientations, respectively; TMV-V-ATPase-S and TMV-V-ATPase-AS  =  the V-ATPase RNA fragment inserted into TMV in sense and antisense orientations, respectively. Numbers indicated with the same letter are homogenous groups at p<0.05.