Rice false smut (RFS) caused by Ustilaginoidea virens is one of the most important diseases in the majority of rice-growing areas worldwide. Rice false smut causes not only yield loss, but also threatens human or animal health by producing cyclopeptide mycotoxins. Cultivar resistance is the most economical, effective and environmentally friendly approach to control RFS. However, development of RFS-resistant rice cultivar still faces big challenges. In the field, disease severity of RFS is largely affected by rice growth period and variable weather conditions. To date, quite a few cultivars with stable resistance to RFS have been identified and could be used as resistant resource for disease resistance breeding (Sun et al., 2020).

In recent years, a RNAi-based approach called host-induced gene silencing (HIGS) has been increasingly developed to control fungal diseases, in which small interference RNAs (siRNAs) that match important genes of the invading pathogen are produced by transgenic host plants to silence fungal genes during infection (Dou et al., 2020; Wang et al., 2020). Here, we ascertained the potential of HIGS for generation of transgenic rice plants against RFS by targeted silencing of three fungal-specific genes of U. virens.

Selection of effective target genes is the key step for RNA silencing in HIGS. At this time, a limited number of virulence genes have been identified in U. virens (Sun et al., 2020). Among them, fungal-specific transcription factors UvCom1 and UvPro1 play a critical role in development and virulence (Chen et al., 2020; Lv et al., 2016). To further reduce the risk of changes in expression of homologous non-target genes in animals or plants, we attempted to use these two genes UvCom1 and UvPro1 and a newly identified fungal-specific septin gene UvAspE (Uv8b_1773) to develop transgenic rice cultivars with RFS resistance. Deletion of UvAspE caused severe defects in hyphal growth and virulence of U. virens (Figure 1a–e). Moreover, UvAspE was localized in septum and cytoplasm (Figure 1f), and deletion of UvAspE caused significantly reduced septum thickness of U. virens (Figure 1g), suggesting that UvAspE is required for fungal development and virulence. Thus, these three fungal-specific virulence genes in U. virens were used as targets in HIGS.

To design RNAi constructs that could silence the three genes (Figure 1h), a DNA fragment containing a 457-bp partial UvAspE-coding region, a 394-bp partial UvCom1-coding region or a 424-bp partial UvPro1-coding region (Figure 1i) was individually inserted into RNAi vector ds1301 to generate a dsRNA sequence with a hairpin structure. The three gene RNAi vectors and empty vector (EV) were then bombarded into japonica rice cultivar Nipponbare (Nip) to generate transgenic plants. There were no noticeable defects in agronomic traits of transgenic plants expressing any of the RNAi constructs. Integration and expression of the RNAi cassettes were verified by PCR and RT-PCR, respectively, in two independent transgenic lines (Figure 1j). After injection with mycelial/conidial suspensions of U. virens wild-type strain HWD-2 at 30 days post inoculation (dpi), resistance of the transgenic rice lines to U. virens was significantly enhanced when compared with those of control plants (Figure 1k,l). Under a JEOL JSM-6390LV scanning electron microscope (SEM), at 3 dpi, hyphae were found to be elongated and extended along the surface of spikelets in Nip and all tested transgenic rice lines. At 6 dpi, abundant hyphae were observed on the surfaces of filaments of Nip, whereas rare hyphae were found on the surface of filaments of the transgenic rice lines (Figure 1m,n). These results revealed that transgenic rice lines could prevent RFS by inhibiting the extension of infection hyphae.

We quantified the transcription level of the three genes in U. virens during the infection process on rice spikelets of T2 transgenic rice plants and Nip at 3 dpi. Relative transcriptional levels of these three genes were all significantly reduced in transgenic lines compared with Nip (Figure 1o). Small RNA sequencing was performed to identify siRNAs specific to the RNAi cassette in transgenic rice lines L2. Sequencing data showed that siRNAs mapping to UvAspE, UvCom1 or UvPro1 were significantly enriched in their respective transgenic rice line, accounting for 0.06% (UvAspE), 0.03% (UvCom1) or 0.05% (UvPro1) of the total small RNAs detected. The lengths of siRNAs mapped to any of the three genes in their transgenic lines were distributed between 18- and 30-bp, and 21-bp siRNA was the most abundant (Figure 1p). In fluorescence in situ hybridization (FISH) assays, fluorescence signal was both observed in rice flower organ and infection hyphae of U. virens in the infected UvCom1RNAi, UvPro1RNAi and UvAspERNAi transgenic rice plants at 6 dpi, while no fluorescence signal was detected in Nip plants (Figure 1q–s). These results demonstrated that RNAi constructs of these three target genes were successfully processed into siRNA molecules in transgenic rice plants, and these siRNAs were translocated to fungal cells during infection, thereby reducing the transcript levels of the three genes in the invading hyphae of U. virens.

Taken together, our results suggest that HIGS targeting the fungal-specific virulence genes in U. virens can be used as an effective approach for developing RFS-resistant rice plants.

由黑粉虱引起的水稻黑穗病（RFS）是全球大部分水稻种植区最重要的病害之一。稻曲病不仅造成产量损失，而且通过产生环肽真菌毒素威胁人类或动物的健康。品种抗性是控制RFS最经济、有效和环保的方法。然而，抗RFS水稻品种的开发仍面临很大挑战。在田间，RFS 的病害严重程度在很大程度上受水稻生育期和多变的天气条件的影响。迄今为止，已鉴定出不少对RFS具有稳定抗性的品种，可作为抗病育种的抗性资源（Sun等，2020）。

近年来，一种称为宿主诱导基因沉默 (HIGS) 的基于 RNAi 的方法越来越多地用于控制真菌疾病，其中与入侵病原体的重要基因相匹配的小干扰 RNA (siRNA) 由转基因宿主植物产生在感染期间沉默真菌基因（Dou等人，2020 年；Wang等人，2020 年）。在这里，我们通过靶向沉默U. virens的三个真菌特异性基因来确定 HIGS 产生针对 RFS 的转基因水稻植物的潜力。

选择有效的靶基因是 HIGS 中 RNA 沉默的关键步骤。目前，已在U.virens中鉴定出数量有限的毒力基因（Sun et al ., 2020）。其中，真菌特异性转录因子UvCom1和UvPro1在发育和毒力中发挥关键作用（Chen et al ., 2020 ; Lv et al ., 2016）。为了进一步降低动物或植物中同源非靶基因表达变化的风险，我们尝试使用这两个基因UvCom1和UvPro1以及新发现的真菌特异性 septin 基因UvAspE ( Uv8b_1773 ) 开发具有RFS抗性的转基因水稻品种。UvAspE的缺失导致U. virens菌丝生长和毒力的严重缺陷（图 1a-e）。此外，UvAspE定位于隔膜和细胞质中（图 1f），并且 UvAspE 的缺失导致U.virens的隔膜厚度显着降低（图 1g），表明UvAspE是真菌发育和毒力所必需的。因此，U.virens 中的这三种真菌特异性毒力基因被用作 HIGS 中的靶标。

为了设计可以沉默三个基因的 RNAi 构建体（图 1h），包含 457-bp 部分UvAspE编码区、394-bp 部分UvCom1编码区或 424-bp 部分UvPro1的 DNA 片段编码区（图 1i）单独插入 RNAi 载体 ds1301 以生成具有发夹结构的 dsRNA 序列。然后将三个基因RNAi载体和空载体（EV）轰击到粳稻品种日本晴（Nip）中以产生转基因植物。表达任何 RNAi 构建体的转基因植物的农艺性状没有明显缺陷。在两个独立的转基因系中，分别通过 PCR 和 RT-PCR 验证了 RNAi 盒的整合和表达（图 1j）。在接种后 30 天 (dpi) 注射U. virens野生型菌株 HWD-2 的菌丝体/分生孢子悬浮液后，转基因水稻品系对U.virens的抗性与对照植物相比，显着增强（图1k，l）。在 JEOL JSM-6390LV 扫描电子显微镜 (SEM) 下，在 3 dpi 处，发现菌丝在 Nip 和所有测试的转基因水稻品系中沿着小穗表面拉长和延伸。在 6 dpi 时，在 Nip 的细丝表面观察到丰富的菌丝，而在转基因水稻品系的细丝表面发现了稀有的菌丝（图 1m，n）。这些结果表明，转基因水稻品系可以通过抑制感染菌丝的延伸来预防 RFS。

我们在 3 dpi 时对 T2 转基因水稻植物和 Nip 的水稻小穗感染过程中U. virens中三个基因的转录水平进行了量化。与 Nip 相比，这三个基因的相对转录水平在转基因系中均显着降低（图 1o）。进行小 RNA 测序以鉴定对转基因水稻系 L2 中的 RNAi 盒特异的 siRNA。测序数据显示，定位到UvAspE、UvCom1或UvPro1的siRNA在各自的转基因水稻品系中显着富集，分别占0.06%（UvAspE）、0.03%（UvCom1）或0.05%（UvPro1 ）。) 检测到的总小 RNA。映射到其转基因系中三个基因中任何一个的 siRNA 的长度分布在 18 和 30 bp 之间，并且 21 bp 的 siRNA 是最丰富的（图 1p）。在荧光原位杂交 (FISH) 测定中，在受感染的 UvCom1 RNAi、UvPro1 RNAi 和 UvAspE 的水稻花器官和U. virens 的感染菌丝中均观察到荧光信号6 dpi 的 RNAi 转基因水稻植株，而在 Nip 植物中未检测到荧光信号（图 1q-s）。这些结果表明，这三个靶基因的 RNAi 构建体在转基因水稻植物中被成功加工成 siRNA 分子，并且这些 siRNA 在感染过程中易位到真菌细胞，从而降低了入侵的U. virens菌丝中三个基因的转录水平。 .

总之，我们的结果表明，针对U. virens中真菌特异性毒力基因的 HIGS可用作开发抗 RFS 水稻植物的有效方法。