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Provoking a silent R gene in wheat genome confers resistance to powdery mildew
Plant Biotechnology Journal ( IF 10.1 ) Pub Date : 2022-07-29 , DOI: 10.1111/pbi.13903
Miaomiao Li 1 , Lei Dong 1, 2 , Keyu Zhu 1, 2 , Qiuhong Wu 1 , Yongxing Chen 1, 2 , Ping Lu 1 , Guanghao Guo 1, 2 , Huaizhi Zhang 1, 2 , Panpan Zhang 1, 2 , Beibei Li 1, 2 , Wenling Li 1, 2 , Yijun Yang 1, 2 , Yikun Hou 1, 2 , Xuejia Cui 1, 2 , Hongjie Li 3 , Lingli Dong 1 , Yusheng Zhao 1, 2 , Zhiyong Liu 1, 2, 4
Affiliation  

Common wheat (Triticum aestivum. L) is one of the most widely cultivated staple crops in the world (Tadesse et al., 2019), and it has been always important to breed pathogen-resistant varieties for safeguarding its production (Singh et al., 2016). However, common wheat genetic diversity has been narrowed throughout its entire existence due to two sequential polyploidization events followed by domestication (IWGSC, 2014), therefore profoundly limiting its improvement like pathogen resistance. Here, we provide an alternative route to increase the genetic diversity toward future wheat breeding by exploiting the silent genetic loci hidden in the wheat genome.

Powdery mildew, caused by the fungus Blumeria graminis f. sp. tritici (Bgt), is a severe foliar disease of wheat causing reduction in grain yield and quality (Savary et al., 2019). Host resistance is widely considered the first and most effective barrier of defence that prevents the invasion of the pathogen (Wu et al., 2021). We previously isolated the powdery mildew resistance gene Pm41 (hereof Pm41a), which encodes a typical coiled-coil, nucleotide-binding site, and leucine-rich repeat protein (CNL) from wild emmer wheat (WEW, Triticum dicoccoides) accession ‘IW2’ (Li et al., 2020). Moreover, three Pm41 haplotypes including Hap1 (Pm41a, ‘IW2’), Hap2 (‘Langdon’, LDN’), and Hap3 (null, ‘Chinese Spring’) were identified in diversified worldwide wheat collection with a Pm41a gene-specific marker WGGB427. To further characterize the natural diversity of Pm41, the entire locus of Pm41 was amplified with overlapping gene-specific primers (Table S1) in a representative diversified panel including 131 WEW, 38 durum wheat (T. durum Desf.), and 31 common wheat (Table S2), resulting in the identification of seven haplotypes (Figure 1a). Pm41a, Hap2, Hap3, and four new haplotypes (Hap4 to Hap7) were identified in the WEW populations (Figures 1b and S1). In contrast, only three haplotypes (Hap2, Hap3, and Hap7) exist in durum and hexaploid wheat populations, suggesting loss of genetic diversity of the Pm41 locus due to domestication and polyploidization bottleneck. Hap2 (hereof Pm41b) only accounts for 5% of the WEW but was predominant in those tested accessions of durum (90%) and hexaploid (90%) wheats (Figure 1b and Table S2). Detailed analysis showed that Pm41b contains an intact Pm41a allelic coding region sequence (Figure S1) but carries two DNA transposons inserted in the promoter and 3′-UTR regions (Figures 1c and S2). Different from the Pm41a allele, which is resistant and well-induced upon Bgt isolate E09 inoculation in the highly resistant WEW accession ‘IW2’ (Figure 1d–f; Li et al., 2020), Pm41b shows no expression before or after Bgt isolate E09 inoculation in the highly susceptible tetraploid durum wheat cultivar ‘LDN’ (Figure 1d–f). Furthermore, a similar result was obtained in the susceptible common wheat cultivar ‘Fielder’ (Figure 1f) carrying the same Pm41b allele as ‘LDN’ (Table S2), suggesting that Pm41b, as well as other Pm41 haplotypes (Figure S2), are silenced alleles of Pm41a, probably due to the transposon insertions and represent unexploited hidden variations for powdery mildew resistance in modern wheat breeding.

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Figure 1
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Provoking a silent Pm41b gene conferring powdery mildew resistance. (a) Haplotype analysis of Pm41 alleles. (b) Genetic bottleneck of Pm41 alleles from wild emmer wheat to domesticated durum and bread wheat. (c) Gene structure and variations of Pm41a and Pm41b. Green, yellow, and red rectangles represent predicted coiled-coil, NB-ARC, and LRR domain, respectively. Black triangles show the nucleotide (amino acid) differences between Pm41a and Pm41b. (d) Leaves at 2-leaf-stage of wild emmer wheat accession ‘IW2’ and durum wheat ‘Langdon’ challenged with Bgt isolate E09 at 14 day post-inoculation (dpi). Scale bar, 3 mm. (e) Fungal structures of Bgt isolate E09 at 5 dpi as stained by Coomassie brilliant blue. AGT, appressorial germ tube; APP, appressorium. Scale bar, 100 μm. (f) RT-PCR analysis of Pm41a and Pm41b in ‘IW2’, ‘Langdon’ and ‘Fielder’. The TdcACTIN, TdACTIN and TaACTIN genes from IW2, Langdon and Fielder, respectively, were used as internal controls. (g) Schematic diagram of ProUbi:Pm41b and ProPm41a:Pm41b used for transformation of powdery mildew susceptible cv. ‘Fielder’. ProPm41a:Pm41b includes a 2388 bp presumed native promoter of Pm41a, the 3370 bp entire genomic sequence of Pm41b including potentially coding and intron regions, and a 1390 bp terminator region of Pm41a. Ubi, promoter of the maize ubiquitin gene. (h) Infection reactions of T1 transgenic plants of ProUbi:Pm41b and (i) ProPm41a:Pm41b. ‘Fielder’ was used as a susceptible control. Three individuals of three independent families are shown. The “+”and “−” signs designate the presence or absence of Pm41b transgenes. (j) The transcript levels of Pm41b in T2 transgenic plants of ProUbi:Pm41b and ProPm41a:Pm41b. Error bars represent ± SEMs of three independent experiments. Statistically significant differences (Student's t-test): **, P < 0.01.

To further explore the functionality of Pm41b in vivo, as well as its possibility of application in wheat breeding, we therefore generated two types of transgenic wheat plants (Ishida et al., 2015) carrying Pm41b driven either by the Pm41a promoter or by the constitutively expressing promoter of ubiquitin from maize (Zea mays L.) (Figure 1g). The T0 and T1 seedlings from both types of transgenic plants were challenged with Bgt isolate E09. The transgenic plants carrying the ubiquitin-driven Pm41b gene were immune to isolate E09 with infection types (ITs) of 0 (immune) - 0; (necrotic fleck) (Figures 1h, S3 and Table S3), while the other transgenic plants carrying Pm41b driven by the native promoter of Pm41a were resistant to isolate E09 with IT of 1 (highly resistant) (Figure 1i and Table S3). Moreover, co-segregation of the Pm41b transgene with powdery mildew resistance was observed in the T2 segregating progenies (Table S3), supporting the functionality of Pm41b in powdery mildew resistance. The transcript level of Pm41b in the transgenic plants carrying the ubiquitin-driven Pm41b gene is about three folds higher than that in the transgenic plants carrying Pm41b driven by the Pm41a native promoter (Figure 1j), in correlation to their resistance, suggesting Pm41b may function in a dosage-dependent way. In addition, the increased powdery mildew resistance of Pm41b-OE and Pm41-COM transgenic plants did not impact on major agronomic traits (Figure S4). The abovementioned results imply that the silent genetic loci like Pm41b are a valuable resource of genetic variation in the wheat genome and therefore could be potentially utilized to enrich the genetic diversity in wheat breeding.

The genomes of wheat, as well as other crops, contain a large proportion of inactive or silenced genetic loci, many of which are related to the key agronomic traits and stress resistance and would enormously contribute to the pool of genetic variation if properly modulated. For instance, about 84% of wheat genome are transposon elements (IWGSC, 2014), exhibiting the significance of provoking those silenced loci due to interference by the transposon elements. In this study, we examined this idea by provoking a silent Pm41b with a functional Pm41a promoter, which surprisingly conferred sound powdery mildew resistance in hexaploid wheat. Our work sheds a light on how to wake up and make use of those ‘sleeping beauties’ in the wheat genome. It provides an intriguing approach to exploit genetic diversity, which is extremely narrowing and being a barrier of crop improvement in modern cultivars. The emergence of precise genome editing tools will offer more efficient approaches to directly modulate these silent but useful loci in modern cultivars and booster the modern crop breeding for food sustainability.



中文翻译:

在小麦基因组中激发沉默的 R 基因赋予了对白粉病的抗性

普通小麦(Triticum aestivum.L)是世界上种植最广泛的主食作物之一(Tadesse et al .,  2019),培育抗病原菌品种以保障其生产一直很重要(Singh et al . ,  2016 年)。然而,由于随后的两次连续多倍化事件以及随后的驯化(IWGSC, 2014 ) ,普通小麦遗传多样性在其整个存在过程中已经缩小 ,因此极大地限制了其改进,如病原体抗性。在这里,我们提供了一种替代途径,通过利用隐藏在小麦基因组中的沉默基因位点来增加未来小麦育种的遗传多样性。

白粉病,由真菌Blumeria graminis f 引起。sp。tritici ( Bgt ) 是小麦的一种严重叶面病害,会导致谷物产量和质量下降(Savary等人,  2019 年)。宿主抗性被广泛认为是防止病原体入侵的第一个也是最有效的防御屏障(Wu et al .,  2021)。我们之前分离了白粉病抗性基因Pm41此处为 Pm41a ),该基因编码来自野生二粒小麦(WEW, Triticum dicoccoides )的典型卷曲螺旋、核苷酸结合位点和富含亮氨酸的重复蛋白(CNL ) 加入 'IW2' (李等人,  2020 年)。此外,通过Pm41a基因特异性标记WGGB427在全球多样化的小麦收集中鉴定了三种Pm41单倍型,包括 Hap1 ( Pm41a , 'IW2')、Hap2 ('Langdon', LDN') 和 Hap3 (null, 'Chinese Spring') . 为了进一步表征 Pm41 的自然多样性,在包括 131 个 WEW、38 个硬粒小麦 ( T. durum Desf.) 和 31 个普通小麦在内的代表性多样化面板中,用重叠的基因特异性引物(表 S1)扩增了Pm41的整个基因座(表 S2),导致鉴定出七种单倍型(图 1a)。pm41a、Hap2、Hap3 和四种新的单倍型(Hap4 到 Hap7)在 WEW 群体中被鉴定出来(图 1b 和 S1)。相比之下,硬粒小麦和六倍体小麦种群中仅存在三种单倍型(Hap2、Hap3 和 Hap7),这表明由于驯化和多倍化瓶颈导致Pm41基因座的遗传多样性丧失。Hap2(此处为Pm41b)仅占 WEW 的 5%,但在那些测试的硬粒小麦(90%)和六倍体(90%)小麦中占主导地位(图 1b 和表 S2)。详细分析显示Pm41b包含完整的Pm41a等位基因编码区序列(图 S1),但携带两个插入启动子和 3'-UTR 区的 DNA 转座子(图 1c 和 S2)。不同于Pm41a等位基因,在Bgt分离株 E09 接种后在高抗性 WEW 加入 'IW2' 中具有抗性和良好诱导(图 1d-f;Li等人,  2020),Pm41b在Bgt分离株 E09 接种之前或之后均未显示表达。高度敏感的四倍体硬粒小麦品种'LDN'(图1d-f)。此外,在携带与“LDN”相同的Pm41b等位基因(表 S2)的易感普通小麦品种“Fielder”(图 1f)中获得了类似的结果,这表明Pm41b以及其他Pm41单倍型(图 S2)是Pm41a的沉默等位基因,可能是由于转座子插入,代表了现代小麦育种中白粉病抗性的未开发的隐藏变异。

详细信息在图片后面的标题中
图1
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激发沉默的Pm41b基因,赋予白粉病抗性。(a) Pm41等位基因的单倍型分析。(b)从野生二粒小麦到驯化硬粒小麦和面包小麦的Pm41等位基因的遗传瓶颈。(c) Pm41aPm41b的基因结构和变异。绿色、黄色和红色矩形分别代表预测的盘绕线圈、NB-ARC 和 LRR 域。黑色三角形显示Pm41aPm41b之间的核苷酸(氨基酸)差异。(d) 用Bgt攻击的野生二粒小麦品种“IW2”和硬粒小麦“Langdon”的 2 叶期叶片在接种后 14 天 (dpi) 分离 E09。比例尺,3 毫米。(e) Bgt分离株 E09 在 5 dpi 时的真菌结构,用考马斯亮蓝染色。AGT,附着胚管;APP,应用程序。比例尺,100 μm。(f) 'IW2'、'Langdon' 和'Fielder'中Pm41aPm41b的RT-PCR 分析。分别来自 IW2、Langdon 和 Fielder的TdcACTINTdACTINTaACTIN基因用作内部对照。(g) Pro Ubi : Pm41b和 Pro Pm41a : Pm41b用于白粉病易感品种转化的示意图。'外野手'。临Pm41aPm41b包括一个 2388 bp 假定的Pm41a天然启动子、一个 3370 bp 的Pm41b完整基因组序列,包括潜在的编码和内含子区域,以及一个 1390 bp 的Pm41a终止子区域。Ubi,玉米泛素基因的启动子。(h) Pro Ubi的 T 1转基因植物的感染反应:Pm41b和 (i) Pro Pm41aPm41b。'Fielder' 被用作易感对照。显示了三个独立家庭的三个人。“+”和“-”符号表示Pm41b转基因的存在或不存在。(j) T 2中Pm41b的转录水平Pro Ubi的转基因植物:Pm41b和Pro Pm41aPm41b。误差条代表三个独立实验的± SEM。统计学显着差异(学生t检验):**,P  < 0.01。

为了进一步探索Pm41b 在体内的功能,以及它在小麦育种中应用的可能性,我们因此生成了两种携带Pm41b的转基因小麦植物 (Ishida et al .,  2015 ),它们由Pm41a启动子或组成型驱动。表达来自玉米(Zea mays L.)的泛素启动子(图 1g)。来自两种类型的转基因植物的T 0和T 1幼苗用Bgt分离物E09攻击。携带泛素驱动的Pm41b的转基因植物基因对分离株 E09 具有免疫性,感染类型 (ITs) 为 0 (免疫) - 0;(坏死斑点)(图1h,S3和表S3),而携带由Pm41a的天然启动子驱动的Pm41b的其他转基因植物对IT为1的分离物E09具有抗性(高度抗性)(图1i和表S3)。此外,在 T 2分离后代中观察到Pm41b转基因与白粉病抗性的共分离(表 S3),这支持了Pm41b在白粉病抗性中的功能。携带泛素驱动的Pm41b的转基因植物中Pm41b的转录水平基因比携带由 Pm41a 天然启动子驱动的Pm41b转基因植物高约三倍(图 1j),与其抗性相关,表明Pm41b可能以剂量依赖性方式起作用。此外, Pm41b-OEPm41-COM转基因植物增加的白粉病抗性对主要农艺性状没有影响(图 S4)。上述结果表明,像Pm41b这样的沉默基因位点是小麦基因组中宝贵的遗传变异资源,因此可以潜在地用于丰富小麦育种中的遗传多样性。

小麦以及其他作物的基因组包含大量不活跃或沉默的基因位点,其中许多与关键的农艺性状和抗逆性有关,如果适当调节,将对遗传变异库做出巨大贡献。例如,大约 84% 的小麦基因组是转座子元件(IWGSC,  2014 年),显示出由于转座子元件的干扰而激发那些沉默的基因座的重要性。在这项研究中,我们通过激发具有功能性Pm41a的沉默Pm41b来检验这个想法启动子,令人惊讶地赋予六倍体小麦良好的白粉病抗性。我们的工作揭示了如何唤醒和利用小麦基因组中的那些“睡美人”。它提供了一种利用遗传多样性的有趣方法,这种方法极其狭窄,是现代品种作物改良的障碍。精确基因组编辑工具的出现将提供更有效的方法来直接调节现代栽培品种中这些沉默但有用的基因座,并促进现代作物育种以实现食品的可持续性。

更新日期:2022-07-29
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