Development of a transgenic rice line with strong and broad resistance against four devastating rice viruses through expressing a single hairpin RNA construct

Rice is a staple food crop feeding nearly half of the world's population. However, rice production is threatened by devastating viral diseases such as rice black-streaked dwarf virus (RBSDV), southern rice black-streaked dwarf virus (SRBSDV), rice stripe virus (RSV) and rice ragged stunt virus (RRSV) (Wang et al., 2022a). Traditional breeding for viral resistance is challenging due to the lack of natural resistance sources (Wang et al., 2022b). The application of RNA interference (RNAi) technology to generate viral resistant transgenic plants provides a promising approach. The expression of hairpin structures of viral sequences by transgenic technology in plants will produce abundant siRNAs targeting viral genomes, and the targeted viral RNA will be degraded by the plant RNAi machinery (Sasaya et al., 2014; tenOever, 2013). In recent years, there have been several reports on producing transgenic rice lines resistant to rice viruses through RNAi (Feng et al., 2021; Zhao et al., 2020). For example, transgenic rice plants expressing an hairpin RNA (hpRNA) targeting the RBSDV S6 gene showed nearly full immunity to RBSDV (Feng et al., 2021). However, conferring resistance against multiple rice viruses in a single transgenic line has not been reported. Theoretically, it is feasible to fuse sequences from multiple viruses to construct a single hairpin structure, thereby conferring resistance against multiple viruses. In this study, we simultaneously targeted four rice viruses, including RBSDV, SRBSDV, RSV and RRSV, by expressing a hairpin RNA construct, and strong and broad resistance against all four viruses was achieved in the transgenic lines. The genomes of RBSDV and SRBSDV both consist of 10 double-stranded RNA (dsRNA) segments, with nucleotide identities exceeding 70%. RRSV also possesses a 10-segment dsRNA genome, but it exhibits significant divergence from those of RBSDV and SRBSDV. In contrast, the genome of RSV consists of four single-stranded RNA (ssRNA) segments, distinguishing it from the aforementioned three viruses (Wang et al., 2022a). We first analysed the encoding sequences of RBSDV P10 protein, SRBSDV P9-1 protein, RRSV P9 protein and RSV-CP protein and cloned 300 bp conserved fragments from each viral protein encoding sequence. These fragments were then fused into a complete sequence. Subsequently, the sense and antisense fused sequences were linked by the intron of Arabidopsis RTM1 gene as a linker, resulting in a chimeric hairpin structure of the fused sequence. This hairpin construct was placed downstream of the maize Ubi-1 promoter (Figure 1a) and inserted into the pCAMBIA1300 vector with the selectable marker replaced by a glyphosate resistance gene. Finally, through Agrobacterium-mediated transformation of rice calli, we obtained a transgenic rice plant line (ZJU-4K) in the background of Wuyunjing 3 (WYJ3). PCR detection of the T4 generation of ZJU-4K plants showed that the inserted hairpin construct could be stably inherited (Figure 1b). Small RNA sequencing of the T4 generation of ZJU-4K plants revealed that the inserted hairpin produced a large number of siRNAs covering the entire hairpin, with the small RNA reads derived from the hairpin accounting for 13.06% of the total reads (Figure 1c). Among them, 21- and 22-nt siRNAs were the most abundant, suggesting their potential vital role in the RNAi-mediated antiviral defence against the targeted viruses in ZJU-4 K rice plants (Figure 1c). To confirm whether the siRNA levels were stable among individual T4 generation of ZJU-4K plants, RT-qPCR analyses of siRNAs from different regions of the hairpin were performed, and the results showed that the siRNA abundance was relatively consistent among sibling plants of T4 generation of ZJU-4K (Figure 1d). Then, the T4 generation of ZJU-4K plants were inoculated with RBSDV, SRBSDV, RSV and RRSV. The rice plants were inoculated with respective viruses through specific planthopper vectors: small brown planthopper (SBPH, Laodelphax striatellus) for RBSDV and RSV, white back planthopper (WBPH, Sogatella furcifera) for SRBSDV and brown planthopper (BPH, Nilaparvata lugens) for RRSV. Each viral inoculation experiment had 3 biological replicates, with at least 30 rice seedlings per replicate. After inoculation, the resistance against the 4 viruses was evaluated by disease incidence, symptoms and viral accumulation. The results showed that T4 generation of ZJU-4K plants exhibited extremely high resistance against the target viruses. The disease incidence of all 4 viruses was significantly lower than the wild-type control (Figure 1e–h). Then the viral protein and nucleic acid accumulation levels were determined through Western blot and RT-qPCR. For RBSDV, SRBSDV and RSV, the results were consistent with disease incidence and symptom observation, with almost no detectable viral accumulation in T4 generation of ZJU-4K plants (Figure 1i–k). However, for RRSV, the viral RNA and protein could still be detected in T4 generation of ZJU-4K plants, but the accumulation was significantly lower than that in the wild-type WYJ3 plants (Figure 1l). Although T4 generation of the ZJU-4K rice plants could not completely resist RRSV infection, it prevented symptom development and damage from RRSV infection. By resequencing the genome of T4 generation of ZJU-4K plants, we confirmed that the T-DNA sequence was inserted as a single copy at an intergenic site on chromosome 1 (between base 23 439 258 and base 23 439 299) (Figure 1m, upper panel). The insertion site was also verified through PCR using specific primers (Figure 1m, lower panel). The plant height, heading date and grain shape between T4 generation of ZJU-4K and wild-type WYJ3 plants were also similar, with the only noticeable difference being a 1-day delay in heading date of ZJU-4K compared to the wild-type WYJ3 plants (Figure 1n). In conclusion, we have developed a transgenic rice plant line with strong and broad resistance against four devastating rice viruses by expressing a long hpRNA construct targeting conserved regions of key viral genes. This provides an effective approach for engineering complex viral resistance in rice and possibly other crops. With appropriate biosafety assessments, our transgenic rice plant line can be utilized to breed elite cultivars for stable rice production. This work was supported by grants from the National Key Research and Development Program of China (2023YFD1400300), the Earmarked Fund for Modern Agro-industry Technology Research System (nycytx-001), the National Natural Science Foundation of China (32372513, 32202260) and the Jiangsu Province Key R&D Program (Modern Agriculture) (BE2022369). The authors thank Prof. Bixia Qin (Guangxi Academy of Agricultural Sciences, Nanning) for RRSV inoculation assistance. The authors have declared no conflict of interest. X.Z. and T.Z. designed the experiments. C.L., J.W., S.F., Y.X., Y.W., X.Y., Y.L., F.L. and D.L. conducted the experiments and analysed the data. C.L. wrote the paper with the input of all other authors. Data sharing is not applicable to this article as no new data were created or analyzed in this study.