Host‐induced gene silencing of Sporisorium scitamineum enhances resistance to smut in sugarcane

Sugarcane smut disease, caused by the basidiomycetous fungus Sporisorium scitamineum, is one of the major threats to the sugarcane industry. The potential method to control smut disease in sugarcane is the cultivation of resistant sugarcane varieties (Bhuiyan et al., 2021). However, development of smut-resistant sugarcane still faces big challenges due to the allopolyploid nature and complex genetic background of sugarcane. To date, functional genomics of sugarcane is far behind those of major crops, for example, wheat and rice, and few genes have been identified that can be used as resistant resources for disease resistance breeding. Host-induced gene silencing (HIGS) technology has become one of the practical methods of developing disease-resistant crops, where transgenic plants produce small interference RNAs (siRNAs) to silence essential pathogen genes (Koch and Wassenegger, 2021). However, application of HIGS to control fungal diseases has not been reported in sugarcane. Here, we assessed the potential of HIGS for engineering sugarcane against smut through silencing of fungal-specific gene of S. scitamineum. Based on our previous transcriptomic results, a fungal-specific gene SsGlcP encoding beta-1,6-glucanase precursor (SPSC_05923) showed induced expression during S. scitamineum infection (Liu et al., 2022). qRT-PCR analysis also confirmed this expression pattern (Figure 1a), suggesting that SsGlcP may play an important role for infection of S. scitamineum. Several studies have suggested the crucial role of β-1,6-glucanase in mycelial growth and development of fungi (Konno and Sakamoto, 2011; Oyama et al., 2006; Wang et al., 2024). To examine whether the exogenous dsRNAs and siRNAs molecules can enter S. scitamineum cells and confer silencing effects, a 564-bp partial SsGlcP coding region (Figure S1) containing several siRNA production sites predicted by siDIRECT 2.0 (http://sidirect2.rnai.jp/) was selected for dsRNA production. Fluorescein-labelled dsRNA was synthesized using fluorescein-12-UTP and then digested into siRNA by RNaseIII (Figure S2). The dsRNA and siRNA were then incubated with haploid sporidium and dikaryotic hyphae of S. scitamineum, respectively. The results indicated a notable presence of fluorescence in both haploid and hyphae following treatment with dsRNA or siRNA, with a more pronounced fluorescence signal observed in samples treated with dsRNA (Figure 1b). qRT-PCR assays revealed that the SsGlcP expression was significantly decreased in both haploid and hyphae following treatment with SsGlcP-dsRNA, whereas treatment using SsGlcP-siRNAs did not significantly reduce SsGlcP expression, similar as the negative controls using eYFP-dsRNA and eYFP-siRNAs (Figure 1c). Thus, we opted to generate an expression construct containing the chimeric hairpin structure to produce the SsGlcP dsRNA (Figure 1d). The construct was introduced into both mating types of S. scitamineum, JG36 (MAT-1) and JG35 (MAT-2). Two transformants from each strain, designated as JG36-SsGlcP-RNAi and JG35-SsGlcP-RNAi, were selected for further analysis. The expression of SsGlcP was significantly down-regulated in all transformed strains compared with the wild-type strains, indicating that there is an intact RNAi system in S. scitamineum and the neck-loop structure can effectively produce dsRNA (Figure 1e). There was no observable defect in mating/filamentation of the SsGlcP silencing strains (Figure 1f). Following the inoculation of sugarcane with S. scitamineum via the acupuncture method, the relative fungal growth was quantified using quantitative PCR. Figure 1g demonstrated that the relative fungal biomass of SsGlcP-RNAi strains was significantly lower than that of the control (JG35 + JG36). To determine whether the virulence of S. scitamineum could be compromised by silencing of SsGlcP gene, we conducted virulence assays using the root dipping method (Lu et al., 2021). The wild-type strains JG35 × JG36 exhibited an average incidence of black whip, a typical symptom of smut, of 67.5% at 65 days post-inoculation, whereas the JG35-SsGlcP-RNAi × JG36-SsGlcP-RNAi strains yielded a whip rate of 6.21% (Figure 1h). Therefore, the SsGlcP gene seems to be appropriate as the target gene for constructing HIGS sugarcane. The chimeric RNAi cassette for sugarcane transformation was generated (Figure 1d) and then introduced into the smut-susceptible sugarcane variety ROC22 using Agrobacterium-mediated transformation. Transgenic sugarcane lines expressing SsGlcP RNAi were confirmed using PCR and RT-PCR (Figure 1i). A total of 66 positive sugarcane transgenic seedlings, referred to as HIGS-SsGlcP lines, were inoculated with S. scitamineum by the root dipping method. The first black whip appeared in wild-type sugarcane (WT) at 32 days post-inoculation, while the first black whip was first observed at 51 days post-inoculation in the HIGS-SsGlcP lines. Histopathological examination revealed the presence of fungal hyphae in the growing point of all sugarcane plants inoculated S. scitamineum (Figure 1j). After 65 days of inoculation, the whip rate in WT, empty vector control lines (EV) and HIGS-SsGlcP lines were 74/134 (56%), 39/75 (52%) and 22/66 (33%), respectively (Figure 1k). To determine the SsGlcP transcript level, six HIGS-SsGlcP lines were randomly selected for qRT-PCR analysis. Figure 1l showed a significant reduction in the transcriptional level of SsGlcP compared to the WT, suggesting sugarcane smut resistance in these transgenic lines was caused by in planta-derived silencing of the target gene. Furthermore, two HIGS-SsGlcP lines were used for agronomic trait analysis, and the results showed that they were indistinguishable from the WT. The natural smut incidences of HIGS-SsGlcP and the expression levels of the SsGlcP gene were lower than those in WT (Figure 1m,n, Table S2). Moreover, the relative fungal biomass of S. scitamineum in infected HIGS-SsGlcP-17 was significantly reduced compared to that of WT (Figure 1o). In conclusion, our findings provide evidence that the application of HIGS targeting the ScGlcP gene of S. scitamineum has the potential to enhance the resistance of sugarcane against smut disease. Consequently, the development of HIGS sugarcane demonstrates a feasibility of targeting an essential gene of the smut fungus as a promising strategy for enhancing smut resistance in sugarcane breeding. This work was supported by National Natural Science Foundation of China (32072408), Guangxi Key Technologies R&D Program (GKAB23026083), Guangxi Science and Technology Major Program (GKAA24206010), State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources (SKLCUSA-a202208), and Academy of Sugarcane and Sugar Industry (ASSI-2022008). The authors declare no conflict of interest. HW, RL and BC designed the research and wrote the manuscript. HW, JQ, PZ and XH performed the experiments and analysed the data. SL and JM provided technical support. All authors have read and approved the final manuscript. The data that supports the findings of this study are available in the supplementary material of this article. Figure S1 Sequence representation of SsGlcP RNAi fragments to produce dsRNA. Figure S2 Analysis of SsGlcP-dsRNA and SsGlcP-siRNA. Table S1 Primers used in this study. Table S2 Performance of the major agronomic traits of HIGS-SsGlcP transgenic sugarcane lines. 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.