Development of marker‐free transgenic rice exhibiting stable and enhanced resistance to Rice ragged stunt virus and Rice grassy stunt virus via RNA interference

Rice is the staple food for approximately half the global population, and rice virus diseases pose a serious threat to rice production, leading to significant losses in yield and quality, endangering agricultural security. Over the past two decades, significant advancements have been made by Chinese scientists in plant virology, encompassing basic research and the development of control measures for plant viral diseases (Wu et al., 2022, 2023). Since the initial identification of rice diseases in 1895, more than twenty viruses have been reported to impair rice yields. Notably, both RRSV and RGSV are transmitted by brown planthoppers, and infection can cause severe dwarfism, reduced fertility in rice plants, drastically affecting grain yields and posing a threat to food security. To date, only two rice virus resistance genes, STV11 and OsAP47, have been cloned and functionally validated (Wang et al., 2014, 2022). As a result, the breeding and development of resistant varieties are crucial. During the genetic transformation of plants, selecting marker genes that confer resistance to antibiotics or herbicides can help introduce economically valuable traits into crops. However, biosafety authorities and consumers are concerned that the selective marker genes may transfer from transgenic crops to the environment, potentially inadvertently conferring resistance to herbicides or antibiotics on weeds or pathogenic microorganisms. Consequently, the development of marker-free transgenic plants has become a focal point in the safety research of genetically modified organisms. The double T-DNA vector transformation method not only retains the desired genes in plant cells but also separates out the resistance genes, thereby obtaining safer transgenic plants. Currently, RNAi technology, used in combination with the double T-DNA vector transformation, has been primarily applied to single virus strains with few studies reporting its use against multiple viruses at once. Considering the prevalence of viral co-infections in agriculture and the fact that different viruses often share the same vectors for transmission, this study has constructed a dual-action RNAi vector without selectable markers. This novel vector was inserted into the Huanghuazhan (HHZ, late-maturing, high-yielding indica rice cultivar) to assess its resistance to multiple diseases and impact on agronomic performance. To construct RNAi expression vectors for different viral segments, we first amplified a 262 bp fragment of the RRSV Pns10 gene and a 271 bp fragment of the RGSV PC5 gene and then fused them to form a 533 bp recombinant fragment (Figure 1a). This recombinant fragment was inserted into the intermediate vector pUCC and then integrated into the plant expression vector pMF-Ubi to form the composite vector pMF-Ubi-(Pns10 + PC5)-RNAi (Figure 1b). The RNAi structure and hygromycin resistance gene HPT were located in two independent T-DNA regions. After sequencing and validation, the recombinant vector was used for rice genetic transformation. Ultimately, we identified 21 T0 transgenic lines at the DNA level, of which 16 lines simultaneously carried the RNAi structure and HPT gene, with a co-transformation rate of 76.19% (Figure S1a). We selected offspring from two positive transformants (#3 and #8) for further segregation and screened for material containing the RNAi structure but lacking the HPT gene. Ultimately, we obtained marker-free transgenic rice in the T3 generation, which stably inherited the (Pns10 + PC5)-RNAi structure without resistance genes (Figure S1b–d). To verify the correct transcription of RNAi structures and the generation of siRNA in the introduced materials, we used RT-PCR to evaluate the expression of RNAi structures in different transformants without virus inoculation. The results showed that the RRSV Pns10 and RGSV PC5 gene fragments can be transcribed normally in transgenic lines 3–1 and 8–9 (Figure S2a,b). In addition, small RNA northern blot analysis confirmed the accumulation of corresponding siRNAs in these two transformants (Figure 1c). This indicates that marker-free transgenic rice had correctly transcribed RNAi structures and produced specific siRNAs before inoculation. We inoculated T3 generation marker-free transgenic rice with RRSV and RGSV and evaluated their virus resistance. Two and 6 weeks after virus inoculation, the HHZ plants exhibited typical severe symptoms of viral infection, while marker-free transgenic rice showed strong and stable resistance to viruses without obvious symptoms (Figure 1d–f). At 2 weeks of inoculation, the virus infection rates of marker-free transgenic rice were 1.70% (RRSV) and 4.07% (RGSV), respectively, significantly lower than those of HHZ at 31.41% (RRSV) and 48.1% (RGSV). Six weeks after virus inoculation, the virus infection rates of the transformants remained below 3.35% (RRSV) and 4.68% (RGSV), while the disease incidence in wild type soared to 82.7% (RRSV) and 55.5% (RGSV) (Figure 1g,h). Virus titres were detected in marker-free transgenic rice and were found to be significantly lower than that of HHZ plants infected with either RRSV or RGSV (Figure 1i,j). In the field trials of artificially inoculated viruses, we evaluated the resistance of T3 generation marker-free transgenic rice to RRSV and RGSV under field conditions. In addition to virus resistance, agronomic traits were also evaluated. The results showed that compared with HHZ, the marker-free transgenic lines exhibited strong resistance to both viruses (Figure 1k,l), confirming the effectiveness of RNAi constructs in field environments. In evaluating the agronomic traits of marker-free transgenic rice, we randomly selected 10 plants from each transgenic subpopulation of the T3 generation. The results showed that the plant height (Figure 1m,r), primary branch number per main panicle (Figures 1n and S3a), grain produced per main panicle (Figures 1o and S3b), tiller number (Figure 1p) and thousand grain weight (Figure 1q) of the marker-free transgenic rice were no significantly different from those of HHZ plants and that the growth period of transgenic rice was basically consistent with the HHZ plants. In summary, the bivalent RNAi marker-free transgenic rice we have developed effectively and continuously enhances resistance to RRSV and RGSV, without significantly affecting yield and offering higher biosafety. These achievements provide substantial material support for future research into rice virus resistance and the innovation of germplasm resources. We thank Professor Shimin Zuo of Yangzhou University for the kind gift of the dual T-DNA expression vector. This work was supported by the National Key Research and Development Program of China (2023YFF1000500), the National Natural Science Foundation of China (Nos 32025031, 32072381). The authors have declared no conflict of interest. S.Z. and J.W. designed the experiments. H.X., P.G., S.L., T.T., C.L., C.C., X.G., Z.Z., Y.Y., X.H. and J.H. conducted the experiments and analysed the data. S.Z. wrote the paper with the input of all other authors. All data and resources supporting the findings of this study are available from the corresponding author, Jian-Guo Wu, upon request. Figure S1 PCR detection of the Hairpin and HYG genes in transgenic rice. Figure S2 RT-PCR analysis of the expression level of viral gene fragments. Figure S3 yield of marker-free RNAi transgenic rice. Table S1 Oligonucleotides Used in This Study Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.