Introduction

Verticillium wilt (VW) is a soil-borne fungus disease in a wide range of plant species including alfalfa and caused yield loss up to 50% in less than 3 years due to the rapid spreading of the pathogen in soil (Vandemark et al., 2006). However, the genes responsible for resistance against V. alfalfae is still unknown. In tomato, a well-studied gene locus for VW resistance is known as Ve (Fradin et al., 2011). However, the orthologues in M. truncatula and M. sativa share low sequence identity with the tomato Ve1 and were not involved in the response to VW (Toueni et al., 2016), indicating a different mechanism of VW resistance in Medicago.

In our previous study, we reported several markers associated with VW resistance in alfalfa. One of them was located on chromosome 8 and showed major effect on VW resistance (Yu et al., 2017). In the present study, we identified two candidate genes, MsVR38 and MsVR39 localized at the same locus and linked each other in a head-to-tail orientation. The gap between these two genes was < 3 kb (Figure 1a). The genomic sequence of MsVR38 was 8.4 kb, which was 1.2 kb longer than MsVR39. Both MsVR38 and MsVR39 contained five exons (Figure 1b). Annotation of MsVR38 and MsVR39 found that both were members of the Toll/Interleukin1 receptor–nucleotide binding site–leucine-rich repeat (TIR-NBS-LRR) gene family, and they both contained 14 LRR repeats (Figure 1c). The alignment of protein sequences showed nearly 90% identity between MsVR38 and MsVR39 (Figure 1d), while < 27% identity was found when comparing them with tomato Ve1 (Figure 1e). We are interested in learning if both MsVR38 and MsVR39 play the same role in response to the VW disease in alfalfa.

To assess the functions of MsVR38 and MsVR39 on VW resistance, we selected resistant and susceptible individuals, R384 and S371, respectively, from 317 alfalfa breeding lines and performed bioassay using V. alfalfae. The responses of plants to V. alfalfae showed that R384 had greater resistance compared with S371 (Figure 1f). We cloned and compared CDS region of MsVR38 and MsVR39 between S371 and R384. A missing nucleotide was identified in MsVR38 of R384, which means MsVR38 in R384 was disrupted and not expressed normally (Figure 1g). Then, we inoculated alfalfa leaves of S371 and R384 with Czapek-Dox medium containing exoproteins from V. alfalfae to analyse gene expression in response to the infection of V. alfalfae. The leaves from S371 displayed more severely wilt syndrome compared with those from R384 after inoculation (Figure 1h). Transcriptomic results suggested that, comparing to MsVR38, the expression of MsVR39 was significantly increased in both S371 and R384, implying the positive role of MsVR39 against V. alfalfae (Figure 1i). This result was further confirmed by qPCR (Figure 1j). The relative expression level of MsVR39 in the control of R384 was even higher than that in S371 after inoculation, which could be one of reasons that render R384 greater resistant than S371.

Next, we identified MtVR130 and MtVR140, the homologs of MsVR38 and MsVR39, respectively, from the M. truncatula genome, a model plant of close relative to M. sativa to further verify their function on VW resistance. Comparable to M. sativa, MtVR130 and MtVR140 were also localized on chromosome 8 with the distance less than 3 kb between each other. More importantly, the pair of R genes are highly identical in M. sativa and M. truncatula (Figure 1k). MtVR130 shared 96% similarity with MsVR38, and MtVR140 shared 93% similarity with MsVR39, implying the conservative function of the R gene pair between the two species.

Wildtype M. truncatula R108 and two Tnt1 insertion mutants: Mtvr130 and Mtvr140 were used for further function assessment. The phenotype of infected plants demonstrated that Mtvr130 was more resistant, whereas Mtvr140 was more susceptible to VW compared with the wildtype R108, suggesting that disruption of MtVR130 increased the resistance to V. alfalfae in M. truncatula. (Figure 1l). The results of fungal recovery assay also demonstrated a higher resistance of Mtvr130 (Figure 1m). Collectively, our results revealed that MtVR130 plays negative role in regulation of the disease response, whereas MtVR140 is a positive R gene on defencing against Verticillium.

A question raised by above results is why the pair of R genes shares high identity but functioned differently on defencing against Verticillium. Earlier studies suggested that self-association of TIR domain is essential for the function of resistance signalling (Zhang et al., 2017). It would be interesting to know whether the TIR association exists between MsVR38 and MsVR39 because of their high-identity sequences (Figure 1n). To answer the question, we cloned the TIR coding sequences from MsVR38 and MsVR39 and ligated them into pDEST32 (bait) and pDEST22 (prey) plasmids for interaction analysis using yeast two hybrid. The results demonstrated that either the TIR domains in MsVR38 or MsVR39 could self-associate to form homodimers. However, the interaction between TIR from MsVR38 and MsVR39 was stronger than those of homodimers, indicating that when MsVR38 and MsVR39 both normally expressed, MsVR38 and MsVR39 were more inclined to form into heterodimers, which could negatively regulate the response of Verticillium wilt resistance (Figure 1o). The discrepancy of binding affinity might be resulted from the major unaligned region in TIR domains of MsVR38 and MsVR39 as shown in Figure 1n.

Plant R genes were considered to confer resistance against pathogens, only few of them govern the susceptibility to plant disease (Lorang et al., 2007). In this study, we found that one of the TIR-NBS-LRR genes, MsVR39 responded positively to VW in M. sativa, while negative effect was observed on the other gene MsVR38. Further investigation on the M. truncatula mutants showed that knockout of MtVR130, the homologue of MsVR38 provided a greater resistance against VW compared with the wildtype. Conversely, mutant of MtVR140, the homologue of MsVR39 was more susceptible. A possible mechanism is that when the pair of highly identical TIR-NBS-LRR genes are both normally expressed, they are more likely to form heterodimers, which inhibits the formation of MsVR39 homodimers, thus causing susceptibility to VW disease.

中文翻译：

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两个相关的抗性基因在苜蓿黄萎病的防御中发挥不同的作用

介绍

黄萎病 (VW) 是一种土壤传播的真菌病害，在包括紫花苜蓿在内的多种植物物种中，由于病原体在土壤中的快速传播，在不到 3 年的时间内导致产量损失高达 50% (Vandemark et al ., 2006 年）。然而，负责抗紫花苜蓿的基因仍然未知。在番茄中，一个经过充分研究的 VW 抗性基因位点被称为Ve（Fradin等人，2011 年）。然而，M. truncatula和M. sativa中的直系同源物与番茄 Ve1 的序列同一性较低，并且不参与对 VW 的响应 (Toueni et al ., 2016)，表明紫花苜蓿中 VW 抗性的不同机制。

在我们之前的研究中，我们报道了几个与紫花苜蓿 VW 抗性相关的标志物。其中一个位于 8 号染色体上，对 VW 抗性表现出重大影响（Yu et al ., 2017）。在本研究中，我们确定了两个候选基因，MsVR38和MsVR39位于同一位点，并以头对尾方向相互连接。这两个基因之间的差距是< 3 kb（图1a）。MsVR38的基因组序列为8.4 kb，比MsVR39长1.2 kb 。MsVR38和MsVR39都含有五个外显子（图 1b）。对 MsVR38 和 MsVR39 的注解发现两者都是Toll/Interleukin1 受体-核苷酸结合位点-富含亮氨酸的重复( TIR-NBS-LRR ) 基因家族，它们都包含 14 个 LRR 重复（图 1c）。蛋白质序列的比对显示 MsVR38 和 MsVR39 之间接近 90% 的同一性（图 1d），而在将它们与番茄 Ve1 进行比较时发现 <27% 的同一性（图 1e）。我们有兴趣了解 MsVR38 和 MsVR39 在响应紫花苜蓿 VW 病时是否发挥相同的作用。

为了评估MsVR38和MsVR39对大众抗性的功能，我们分别从 317 个苜蓿育种系中选择了抗性和易感个体 R384 和 S371，并使用V. alfae进行了生物测定。植物对紫花苜蓿的反应表明，与 S371 相比， R384具有更大的抗性（图 1f）。我们在 S371 和R384之间克隆并比较了MsVR38和MsVR39 的 CDS 区域。在R384的 MsVR38中发现了一个缺失的核苷酸，这意味着MsVR38在 R384 中被破坏并且不正常表达（图 1g）。然后，我们用含有来自V的外蛋白的 Czapek-Dox 培养基接种 S371 和 R384 的苜蓿叶。 苜蓿分析响应V感染的基因表达。 苜蓿。与接种后的 R384 相比，S371 的叶子表现出更严重的枯萎综合征（图 1h）。转录组学结果表明，与MsVR38相比，S371 和 R384 中MsVR39的表达均显着增加，这表明MsVR39对V. alfalfae具有积极作用（图 1i）。qPCR 进一步证实了这一结果（图 1j）。接种后MsVR39在R384对照中的相对表达水平甚至高于S371，这可能是R384比S371抗性更强的原因之一。

接下来，我们分别从M中识别出MsVR38和MsVR39的同源物MtVR130和MtVR140。 truncatula基因组，一种与M. sativa近缘的模式植物，以进一步验证它们对 VW 抗性的功能。与M. sativa相比，MtVR130和MtVR140也位于第 8 号染色体上，彼此之间的距离小于 3 kb。更重要的是，这对 R 基因在M. sativa和M中高度相同。 截形（图 1k）。MtVR130与MsVR38的相似度为96%，而MtVR140与MsVR39的相似度为93%，表明R基因对在两个物种之间具有保守功能。

野生型M. truncatula R108 和两个Tnt1插入突变体：Mtvr130和Mtvr140用于进一步的功能评估。受感染植物的表型表明，Mtvr130的抗性更强，而与野生型 R108 相比， Mtvr140对 VW 更敏感，这表明MtVR130的破坏增加了蒺藜苜蓿对苜蓿的抗性。（图 1l）。真菌回收试验的结果也表明Mtvr130具有更高的抗性（图 1m）。总的来说，我们的结果表明MtVR130MtVR140是抗黄萎病的阳性 R 基因。

上述结果提出的一个问题是，为什么这对 R 基因具有高度同一性，但在防御黄萎病菌时的功能却不同。早期的研究表明，TIR 结构域的自关联对于抗性信号传导的功能至关重要（Zhang et al ., 2017）。了解 MsVR38 和 MsVR39 之间是否存在 TIR 关联会很有趣，因为它们具有高同一性序列（图 1n）。为了回答这个问题，我们从MsVR38和MsVR39中克隆了 TIR 编码序列并将它们连接到pDEST32（诱饵）和pDEST22（猎物）质粒中，使用酵母两种杂交体进行相互作用分析。结果表明，MsVR38 或 MsVR39 中的 TIR 结构域可以自缔合形成同源二聚体。然而，来自MsVR38和MsVR39的TIR之间的相互作用强于同源二聚体，表明当MsVR38和MsVR39都正常表达时，MsVR38和MsVR39更倾向于形成异源二聚体，从而负调控黄萎病抗性反应（图1）。 1o)。如图 1n 所示，结合亲和力的差异可能是由于 MsVR38 和 MsVR39 的 TIR 结构域中的主要未对齐区域造成的。

植物 R 基因被认为赋予对病原体的抗性，其中只有少数控制植物病害的易感性（Lorang等，2007）。在这项研究中，我们发现 TIR-NBS-LRR 基因之一 MsVR39对M. sativa中的 VW 有积极反应，而对另一个基因MsVR38观察到负面影响。对M. truncatula突变体的进一步研究表明，与野生型相比，MtVR130（MsVR38 的同源物）的敲除提供了对VW 的更大抗性。相反，MtVR140 的突变体，MsVR39的同源物更容易受到影响。一个可能的机制是，当这对高度相同的TIR-NBS-LRR基因都正常表达时，它们更有可能形成异二聚体，从而抑制MsVR39同源二聚体的形成，从而导致对VW病的易感性。