Soybean cyst nematode (SCN), Heterodera glycines, is a devastating pathogen in soybean worldwide, causing huge yield losses annually. Host-induced gene silencing (HIGS) has shown potentials to control plant parasitic nematodes by generating transgenic plants carrying hairpin constructs against nematode target genes (Chaudhary et al., 2019; Li et al., 2010; Mani et al., 2020; Shivakumara et al., 2017; Tian et al., 2016). Chitin synthesized by chitin synthase (Chs) is present in fungi and nematodes but absent in plants and vertebrate animals and is a target in Magnaporthe oryzae and Fusarium graminearum for designing new fungicides and developing novel resistant varieties by HIGS (Cheng et al., 2015; Kong et al., 2012). Here, we mainly aimed to develop heritable SCN CHS-HIGS soybeans conferring enhanced SCN resistance. Meanwhile, the developed HIGS soybeans were tested for resistance to fungus Fusarium oxysporum, which causes soybean Fusarium wilt disease.

First, the sole CHS gene (SCN-CHS) with 3984 bp (GenBank Acc. No.: OK149168) was cloned from SCN HG Type 1.2.3.5.7 (race 4, SCN4). SCN-Chs contained a typical chitin synthase catalytic domain (Chs) and seven transmembrane domains (TMs) (Figure 1a). qRT-PCR analysis indicated that SCN-CHS was highly expressed at egg stage when compared to other developmental stages (Figure 1b). A 420 bp cDNA fragment of SCN-CHS catalytic region positioned at 1936–2355 bp was used to generate transgenic HIGS soybeans employing ‘Jack’ as wild-type soybean (Figure 1c), and three homozygous transgenic lines (‘48-7-5’, ‘55-8-24’, and ‘57-9-2’) with yellow seed-coat identical to that of Jack (Figure 1d) were obtained. The amount of SCN4 cysts per plant (left of Figure 1e) and eggs per cyst (Figure 1f) in T2 lines was all significantly reduced when compared to Jack. The cyst numbers among T2 lines differed dramatically from 18 to 76, while the cyst numbers were about 120 in Jack, on the average, and the T2 line ‘55-8-24(T2)’ showed the most inhibited SCN cyst formation with a 6-fold reduction in cyst numbers (left of Figure 1e). These results indicated that various HIGS lines suppressed SCN cyst formation differently. The average numbers of eggs per cyst were 153 in T2 lines, which were significantly decreased by 37.5% when compared to that in Jack (Figure 1f). qRT-PCR analysis showed that SCN-CHS expression in eggs of SCN parasitizing in T2 lines was significantly reduced (Figure 1g). These results suggested that down-regulation of SCN-CHS expression in eggs was likely associated with suppression of SCN cyst and egg formation in T2 soybeans. Analyses of the time-course developmental progress of SCN4 juveniles in T2 roots showed that all juveniles were decreased, and the developmental transition frequency from low to high molt stages was also delayed in all T2 roots (Figure 1h). What is more, the size of cysts formed in T2 lines ranged from 662.33 to 673.92 μm in diameter and was obviously smaller than that in Jack (703.4–724.24 μm) (Figure 1i).

Meanwhile, a similar but stronger effectiveness on the suppression of cyst formation was observed in all the SCN HG Type 0 (race 3, SCN3)-infected T2 lines (right of Figure 1e). These results together with SCN4 infection results implied that T2 HIGS soybean lines expressing dsRNA of SCN-CHS showed enhanced broad-spectrum resistance to different SCN HG types (races).

The heredity of HIGS soybeans suppressing SCN was evaluated using three T6 lines, ‘48-7-5(T6)’, ‘55-8-24(T6)’, and ‘57-9-2 (T6)’, with infection of SCN4. The amount of both cysts per plant and eggs per cyst was also significantly decreased in all T6 lines (Figure 1j). Moreover, expression of SCN-CHS in SCN eggs was also obviously suppressed in all T6 lines (Figure 1g). All these results suggested that T6 HIGS lines exhibited strong heredity of the boosted resistance to SCN.

Subsequently, the three HIGS soybean lines were tested for their resistance against Fusarium oxysporum f. sp. glycines. The longer the lesions are on the hypocotyls, the more serious the disease is. The results showed that the average hypocotyl lesion length in all HIGS lines was significantly decreased when compared to that in Jack (Figure 1k). Afterwards, F. oxysporum was isolated from each HIGS line and Jack as Fo-48-7-5, Fo-55-8-24, Fo-57-9-2, and Fo-Jack, respectively, and they were then cultured on PDA-medium plates and mycelia growth was observed. About 1 week post-inoculation, the average spread size (diameter) of mycelia isolated from all the HIGS soybeans was remarkably reduced when compared to that isolated from Jack (Figure 1l). Meanwhile, after these isolated fungi inoculating Jack, the lesions of hypocotyls infected by Fo-48-7-5, Fo-55-8-24, or Fo-57-9-2 were all significantly shorter than these infected by Fo-Jack (Figure 1m). These results indicated that HIGS of SCN-CHS significantly enhanced soybean resistance against F. oxysporum.

In summary, our study showed that host-induced silencing of chitin synthase gene in SCN (SCN-CHS) broadly and durably enhanced soybean resistance against both different SCN races and F. oxysporum, and the developed SCN-CHS HIGS soybeans will be a potential for controlling SCN and Fusarium wilt diseases.

大豆胞囊线虫（SCN）， Heterodera Gans ，是全世界大豆的一种毁灭性病原体，每年造成巨大的产量损失。宿主诱导的基因沉默（HIGS）已显示出通过产生携带针对线虫靶基因的发夹结构的转基因植物来控制植物寄生线虫的潜力（Chaudhary 等人， 2019 ；Li等人， 2010 ；Mani等人， 2020 ；Shivakumara等人， 2017 ；田等人， 2016 ）。由几丁质合酶（Chs）合成的几丁质存在于真菌和线虫中，但在植物和脊椎动物中不存在，并且是稻瘟病菌和禾谷镰刀菌中用于通过HIGS设计新杀菌剂和开发新抗性品种的靶标（Cheng等， 2015 ； Kong等人， 2012 ）。在这里，我们的主要目标是开发可遗传的 SCN CHS -HIGS 大豆，赋予增强的 SCN 抗性。同时，还测试了开发的 HIGS 大豆对引起大豆枯萎病的尖孢镰刀菌的抗性。

首先，从 SCN HG 1.2.3.5.7 型（种族 4，SCN4）克隆了唯一的CHS基因（ SCN-CHS ），长度为 3984 bp（GenBank 登录号：OK149168）。 SCN-Chs 包含一个典型的几丁质合酶催化结构域 (Chs) 和七个跨膜结构域 (TM) (图 1a)。 qRT-PCR 分析表明，与其他发育阶段相比， SCN-CHS在卵期高度表达（图 1b）。位于 1936-2355 bp 的SCN-CHS催化区的 420 bp cDNA 片段用于生成采用“Jack”作为野生型大豆的转基因 HIGS 大豆（图 1c）和三个纯合转基因品系（“48-7-5”）。 ”、“55-8-24”和“57-9-2”），其黄色种皮与 Jack 相同（图 1d）。与 Jack 相比，T2 品系中每株植物的 SCN4 包囊数量（图 1e 左侧）和每个包囊的卵数量（图 1f）均显着减少。 T2 品系之间的包囊数量差异显着，从 18 到 76 个不等，而 Jack 中的包囊数量平均约为 120 个，T2 品系“55-8-24(T2)”显示出最受抑制的 SCN 包囊形成，囊肿数量减少 6 倍（图 1e 左侧）。这些结果表明，不同的 HIGS 系对 SCN 囊肿形成的抑制作用不同。 T2系中每个包囊的平均卵数为153个，与Jack相比显着减少了37.5%（图1f）。 qRT-PCR分析表明，T2系寄生的SCN卵中SCN-CHS表达显着降低（图1g）。这些结果表明，卵中SCN-CHS表达的下调可能与 T2 大豆中 SCN 包囊和卵形成的抑制有关。 对T2根中SCN4幼虫发育进程的时程分析表明，所有幼虫均减少，并且所有T2根中从低蜕皮阶段到高蜕皮阶段的发育转变频率也延迟（图1h）。更重要的是，T2系形成的包囊直径范围为662.33至673.92μm，明显小于Jack的包囊直径（703.4-724.24μm）（图1i）。

同时，在所有 SCN HG 0 型（种族 3，SCN3）感染的 T2 系中观察到类似但更强的抑制包囊形成的效果（图 1e 右侧）。这些结果与 SCN4 感染结果一起表明表达SCN-CHS dsRNA 的 T2 HIGS 大豆品系对不同 SCN HG 类型（种族）表现出增强的广谱抗性。

使用感染后的三个 T6 品系“48-7-5(T6)”、“55-8-24(T6)”和“57-9-2(T6)”评估抑制 SCN 的 HIGS 大豆的遗传性。 SCN4的。在所有 T6 系中，每株植物的包囊数量和每个包囊的卵量也显着减少（图 1j）。此外，SCN卵中SCN-CHS的表达在所有T6系中也明显受到抑制（图1g）。所有这些结果表明T6 HIGS品系表现出强烈的SCN抗性增强遗传性。

随后，测试了三个 HIGS 大豆品系对尖孢镰刀菌 (Fusarium oxysporum f) 的抗性。 sp。甘氨酸。下胚轴上的病变越长，病情越严重。结果表明，与 Jack 相比，所有 HIGS 系的平均下胚轴损伤长度显着缩短（图 1k）。随后，从各 HIGS 系和 Jack 中分离出尖孢镰刀菌，分别为 Fo-48-7-5、Fo-55-8-24、Fo-57-9-2 和 Fo-Jack，然后进行培养在 PDA 培养基平板上观察到菌丝体生长。接种后约 1 周，与从 Jack 分离的菌丝体相比，从所有 HIGS 大豆分离的菌丝体的平均扩散大小（直径）显着减小（图 1l）。同时，这些分离真菌接种Jack后，Fo-48-7-5、Fo-55-8-24或Fo-57-9-2感染的下胚轴病变均明显短于Fo-Jack感染的下胚轴。 （图1m）。这些结果表明SCN-CHS的HIGS显着增强了大豆对尖孢镰刀菌的抗性。

总之，我们的研究表明，宿主诱导的 SCN 几丁质合酶基因 ( SCN-CHS ) 沉默广泛而持久地增强了大豆对不同 SCN 品种和F 的抗性。 oxysporum和开发的SCN-CHS HIGS 大豆将具有控制 SCN 和镰刀菌枯萎病的潜力。