Leafhoppers

FDp strain FD-PEY05 was used for inoculations. It was transmitted to broad bean (Vicia faba) by S. titanus leafhoppers, collected in 2005 in FD-infected vineyards in Peyrière, South-west France (Papura et al., 2009 (link)). The FD-PEY05 genotype belongs to the map-FD2 genetic cluster, and is widely distributed in the main outbreaks of Western Europe (Arnaud et al., 2007 (link); Papura et al., 2009 (link)). The strain has been maintained since then by serial transmission on broad bean, using Euscelidius variegatus as a vector (Caudwell et al., 1972 ).

Nilaparvata lugens populations at seven rice research centers were included in the study. The colonies were initiated between 2004 and 2012 using wild caught individuals from rice fields located near each research center. The centers, with corresponding locations and years of planthopper collections, were as follows: (1) Directorate of Rice Research (DRR-India): (2010) Hyderabad, Andhra Pradesh, India; (2) Hi-Bred Private Ltd. (Pioneer-India): (2007) Medak, Andhra Pradesh, India; (3) Andhra Pradesh Rice Research Institute (APRRI-India): (2004) West Godavari, Andhra Pradesh, India; (4) Punjab Agricultural University (PAU-India): (2007) Ludhiana, Punjab, India; (5) Chiayi Agricultural Experiment Station (CAES-Taiwan): (2012) Chiayi, Taiwan; (6) Southern Regional Plant Protection Center (SRPPC-Vietnam): (2012) Ling Dinh, Vietnam; (7) International Rice Research Institute (IRRI-Philippines): (2009) Los Baños, Philippines.
We also evaluated resistance against S. furcifera colonies at two East Asian centers: CAES and IRRI. The colonies were initiated with wild-caught individuals collected during the same years and at the same locations as the corresponding N. lugens populations (indicated above). Resistance against a single N. virescens colony, located at IRRI, was also evaluated in the study. The colony was initiated with wild leafhoppers from rice fields in Laguna Province (Philippines) that were collected in 2008.
All colonies (N. lugens, S. furcifera and N. virescens) were initiated with ca. 500 adults placed on the susceptible variety Taichung Native 1 (TN1) (≥30 days after sowing) in wire mesh cages of 120 × 60 × 60 cm (H × W × L) under greenhouse conditions (temperatures ranged from 25 to 45 ^°C, L12:D12 photoperiod). During the first two generations of rearing, the colonies were carefully monitored to eliminate diseased and virus carrying individuals.

The laboratory GRLH colony was founded from individuals collected from the paddy fields at the Zhejiang University experiment farm, Hangzhou, China, in 2014. The paddy fields were set up in 2010 and RDV has never occurred. The colony has been reared and maintained on healthy rice plants (TN1, Taichung Native 1 with GRLH susceptibility at the tillering stage) for 3–4 generations within 80–mesh insect proof cages (50 cm³) in a climate chamber at our standard conditions, 27 ± 1°C, 75 ± 5% relative humidity, a 14 L: 10 D photoperiod and light intensity of 3, 500–4, 000 lux.
To obtain RDV-infected GRLH, non-viruliferous nymphs were confined with RDV-infected TN1 rice seedlings (provided by Prof. Li Yi, College of Life Sciences, Peking University, Beijing, China) for 2 days, then transferred to pass the RDV infection along to TN1 rice seedlings. To ensure the GRLHs were viruliferous, nymphs were individually released into separate glass tubes (D = 2.5 cm × H = 25 cm) with one TN1 rice seedling that was given a reference number. Two days later, each rice seedling was separately transplanted in the glasshouse with its reference number and was replaced with new rice seedlings age 15 ± 2 d for the same leafhopper. Ten days later, the GRLHs were individually collected from plants with characteristic RDV symptoms [15 ]. GRLHs were separately reared on RDV-infected TN1 plants in a glass tube in a separate climate chamber set at our standard conditions. After emergence, one female and one male were mated in an 80-mesh cage (50 cm³) with RDV-infected TN1 plants for oviposition. Offspring were collected for RT-PCR as described below. After confirming infections, other offspring were continually reared together to produce a viruliferous colony for the experiments.

The reproductive organs were individually dissected from the newly emerged male adults of RdFV and RGDV co-positive R. dorsalis population, and the relative transcript levels of clip-domain serine protease genes and PPO were examined by RT-qPCR assays. The male reproductive organs were also examined to determine the conversion of PPO to PO in western blot assays using PPO and histone H3 antibodies (0.5 μg/μl). A pool of 30 RGDV-positive males was used for each replicate in RT-qPCR and western blot assays, respectively. The experiment was conducted in at least three replicates for RT-qPCR and western blot assays. To analyze effect of RGDV infection on PO activity, the reproductive organs dissected from approximate 100 newly emerged males were homogenized with the His-Mg buffer (0.1 M histidine, 0.01 M MgCl₂, pH 6.2) buffer in liquid nitrogen. The supernatant was gently mixed with 1 mM dopamine in 10 mM Tris-HCl buffer (pH 8.0) in a 96-well plate at room temperature for 5 min. Enzyme activity was measured using the phenoloxidase kit (Geruisi, G0146W) according to the manufacturer’s protocol. To analyze the effect of M. luteus infection on PO activity, freeze-dried M. luteus was dissolved in water, and then microinjected in dose of ~23 ng/leafhopper into newly emerged males. At 24-h post microinjection, the reproductive organs of approximate 100 RGDV-infected or M. luteus-treated males were dissected and tested for PO activity.
We then tested the effect of knockdown of PPO or HongrES1 expression on PO activity and RGDV infection. The newly emerged male adults of RdFV and RGDV co-positive R. dorsalis population were microinjected with dsGFP, dsPPO or dsHongrES1 (~200 ng/leafhopper). The male reproductive organs of these tested leafhoppers were individually collected and dissected for RT-qPCR and western blot assays to determine the effect of dsRNAs on the expression levels of HongrES1, PPO, or RGDV P8, and the conversion of PPO to active PO, as well as PO activity. A pool of 30 males was used for each replicate in RT-qPCR and western blot assays, respectively. A pool of 100 males was tested for each replicate in PO activity. The experiment was conducted in three replicates for RT-qPCR and western blot assays, as well as PO activity tests.

RNA inference was performed to knock down the expression of related genes. The T7 promoter with the sequence 5′-ATTCTCTAGAAGCTTAATACGACTCACTATAGGG-3′ was added to the forward and reverse primers at the 5′ terminal to amplify a region of ~500–800 bp of HongrES1, RdFV CP, RGDV P8, or GFP gene (Supplementary Table 1). The PCR products were used for the synthesis of dsRNAs targeting HongrES1 (dsHongrES1), RdFV CP (dsCP), RGDV P8 (dsP8), or GFP (dsGFP) according to the protocol for the T7 RiboMAX Express RNAi System kit (Promega, P1700).
To test the knockdown of HongrES1 expression on RdFV or RGDV infection in male reproductive systems, newly emerged male adults of RdFV-positive or RGDV-positive R. dorsalis population were microinjected with dsHongrES1 or dsGFP (approximately 200 ng/leafhopper) using a Nanoject II Auto-Nanoliter Injector (Spring), and then transferred to healthy rice seedlings. To test the knockdown of RdFV CP or RGDV P8 expression on viral infection and HongrES1 accumulation in male reproductive system, newly emerged male adults of RdFV and RGDV co-positive R. dorsalis population were microinjected with dsCP, dsP8 or dsGFP (~200 ng/leafhopper), and then transferred to rice seedlings. For each treatment, approximate 100 insects were microinjected, and three replicates were performed.
The male reproductive organs were dissected to test the expression levels of RdFV CP, RGDV P8, or HongrES1 using RT-qPCR and western blot assays. A pool of 30 dsRNA-treated males was used for each replicate, and the experiment was conducted in three replicates for RT-qPCR assays. The total proteins from reproductive organs of 30 dsRNA-treated males were analyzed for the protein levels in western blot assays by using HongrES1-, CP- or P8-specific IgG (0.5 μg/μl). Experiment was conducted in three replicates in western blot assays. To determine the effect of dsHongrES1, dsCP or dsP8 treatment on paternal transmission of RdFV or RGDV, one dsHongrES1-, dsCP-, dsP8- or dsGFP-treated RdFV- or RGDV-positive male mated with one virus-free virgin female in a glass tube containing a rice seedling for 3 days (Supplementary Table 3). Ten pairs were performed for each treatment. The males were then tested for presence of RdFV or RGDV, and females were left in the tubes for oviposition. The offspring of each mating combination were tested for presence of RdFV or RGDV by RT-PCR assays. The primers used in RT-PCR assays were shown in Supplementary Table 1. The experiment was conducted in three replicates.

To detect the binding of RdFV CP with sperms in vitro, His-tag-fused CP was expressed in Escherichia coli strain Rosetta, and the proteins were purified using nickel-nitrilotriacetic acid resin (Qiagen). Sperm smears collected from testes of RdFV-free R. dorsalis were successively incubated with the purified CP (0.5 μg/μl) for 1 h, smeared on poly-lysine-treated glass slides, immunolabeled with CP-rhodamine (0.5 μg/μl), stained with DAPI (2.0 μg/ml), and then processed for immunofluorescence microscopy.
In neutralization experiments to test the direct interaction between RdFV CP and HongrES1, mature sperms excised from the testes of RdFV-free R. dorsalis were pre-incubated for 30 min with pre-immune antibody (0.5 μg/μl) or HongrES1 antibody (0.5 μg/μl), and then the in vitro CP-sperm binding experiment was performed as described above.
In neutralization experiments to test the direct interaction between RGDV particles and HongrES1, mature sperms excised from RGDV-free leafhoppers were pre-incubated for 30 min with pre-immune antibody (0.5 μg/μl) or HongrES1 antibody (0.5 μg/μl), incubated with the purified RGDV particles (1.0 μg/μl) for 1 h, smeared on poly-lysine-treated glass slides, immunolabeled with P8-FITC (0.5 μg/μl), stained with DAPI (2.0 μg/ml), and then processed for immunofluorescence microscopy.