Plant Viruses

Total RNA was extracted from virus-infected plants using the Direct-zol RNA MiniPrep Plus (Zymo Research) according to the manufacturer’s recommendations. For each sample, 10–15 μg of RNA was separated on a denaturing 2% agarose gel, blotted on a Hybond-N⁺ (GE Healthcare) membrane and hybridized with a DIG-labelled probe. For virus expression analysis, a DIG-labelled RNA probe was synthesized using DIG Northern Starter Kit (Roche) and manufacturer’s instructions were followed. For crRNA detection, a 5’-end DIG-labelled oligonucleotide (IDT) was used. DIG application manual (Roche) was followed for capillary transfer, hybridization, and detection. Northern blots were repeated in three independent experiments with the same results.

All plants used in the study were grown under greenhouse conditions at the International Institute of Tropical Agriculture (IITA), Dar es Salaam, Tanzania. The varieties of cassava and tomato used were Albert and Moneymaker, respectively, while sweet potato and cotton were local landraces. All greenhouse plants were grown in pots with a soil mix of forest soil and manure mixed in a 4:1 ratio. Plants were typically 20–30 cm tall or with a minimum of five leaves. “Albert” is known to be preferred by whiteflies, while the tomato cultivar—Moneymaker—is extensively used in the scientific literature.
Considering the numerous reports of vector feeding behavior manipulation by viruses, it was important to ensure the use of virus-free cassava plants (Liu et al., 2013 (link); Moreno-Delafuente et al., 2013 (link); Lu et al., 2017 (link)). All cassava planting material was obtained from CMD and CBSD asymptomatic fields in Mtwara Region, Tanzania. Furthermore, leaf samples were taken from each stem and tested for the presence of Cassava Brown Streak Ipomoviruses using real-time RT-PCR to exclude the possibility of asymptomatic infections. The CBSI virus testing was done using the protocol described for cassava by Shirima et al. (2017 (link)). Leaf samples in the form of the middle leaflet were taken from the fifth youngest leaf from each cassava stem cutting used for planting. Samples were dried between two sheets of paper at room temperature for 4 days. Total RNA was extracted using the acetyltrimetyl ammonium bromide (CTAB) protocol, cassava complementary DNA (cDNA) was synthetized and real-time polymerase chain reaction was performed using primers, probes, and cycling conditions described in Shirima et al. (2017 (link)). “Albert” is resistant to CMD, and since only symptomless plants were selected for planting material, the risk of infection was considered low and the material was not tested for the presence of CMBs. The assumption of the absence CMD was further supported as none of the grown plants exhibited the symptoms. Sweet potato plants were asymptomatic. Vegetative material used to plant them has been maintained under insect-proof screenhouse conditions, without symptoms of virus infection, for 3 years. Tomato and cotton were grown from certified seed.

Classification of virus-positive spontaneous plants was based on plant morphology and DNA barcode rbcL and matK genes, according to Fazekas et al. [33 (link)]. First, DNA extraction was done using CTAB method [34 ]. Then, the rbcL and matK genes were amplified with Taq DNA Polymerase, recombinant (Invitrogen, Carlsbad, CA, USA) using the sets of primers SI_For/SI_Rev and KIM 3F/KIM 1R [35 (link)] (Table S1).
All amplicons were visualized by electrophoresis in agarose gel stained with ethidium bromide (Invitrogen, Carlsbad, CA, USA). PCR products were excised from the gel, purified, and Sanger sequenced at Macrogen Inc. (Seoul, Republic of Korea). Sequences were assembled in Geneious Prime^® 2022.1.1. and analyzed using the BOLD Identification System website [36 (link)].

The cDNA sequences of TaELP2 were analyzed using SiFi12 software, and two segments with high and specific siRNA numbers were selected for gene silencing, TaELP2-as1 and TaELP2-as2, respectively. Specific primers (Table S10) were designed, and two specific cDNA fragments of TaELP2 were obtained using PCR and constructed into BSMV:γ genome. Then, the seedlings (Yannong 19) at the two-leaf stage were inoculated using friction. After 10 days of inoculation, the virus phenotype of the leaves was observed. The plants with virus phenotype were identified with RT-qPCR and treated in dark. The leaf phenotype and chlorophyll content were observed at 9 days and 14 days after treatment. The determination method of chlorophyll content was from Porra et al. [84 (link)].

Rice cultivar Oryza sativa L. japonica. Nipponbare (NIP) was used in this study. The cultivar Nipponbare is highly susceptible to SRBSDV and RSV (Xu et al., 2014 (link); Yang et al., 2018b (link)). NIP was used to produce transgenic rice. SRBSDV-infected plants were kindly provided by Professor Guohui Zhou and Tong Zhang (South China Agricultural University Guangzhou, China). Rice seedlings were grown in a greenhouse at 26 to 28°C with a 14-h light/10-h dark cycle under artificial light. Rice plants infected with SRBSDV and RSV were cultivated in an experimental field in Changsha and Nanjing, respectively, under natural long-day conditions. Viruliferous or virus-free planthoppers were reared on healthy rice seedlings (Wuyujing No. 3) in glass beakers at 25°C.
SRBSDV was transmitted by the white backed planthopper (Sogatella furcifera) at approximately the 1.5-leaf-stage of rice seedling. To obtain viruliferous insects, nymphs were reared on virus-infected rice plants for 2 days, and viruliferous or virus-free nymphs were transferred to each experimental rice plant to feed for 3 days, after which the planthoppers were removed. The proportions of healthy plants were calculated 30 days after inoculation. The percentage of about 30 plants infected by virus (viral incidence) of each of triplication was determined following specific quantitative RT−PCR of samples of each plant using virus-specific primers (Table S1) and western blotting using SRBSDV P8 polyclonal antibody.
RSV was transmitted experimentally to rice plants by the small brown planthopper (Laodelphax striatellus) at approximately the 1.5-leaf-stage of rice seedling. To obtain viruliferous insects, nymphs were reared on virus-infected rice plants for 2 days, and viruliferous or virus-free nymphs were transferred to each experimental rice plant to feed for 3 days, after which the planthoppers were removed. The proportions of healthy plants were calculated 30 days after inoculation. The percentage of about 30 plants infected by virus (viral incidence) of each of triplication was determined following specific quantitative RT−PCR of samples of each plant using virus-specific primers (Table S1).