Anton Hochmuth, Matthew Carswell, Aaron Rowland, Danielle Scarbrough, Lara Esch, Nitin Uttam Kamble, Jeffrey W. Habig, David Seung
The molecular mechanisms underpinning the formation of the large, ellipsoidal starch granules of potato tuber are poorly understood. Here, we demonstrate the distinct effects of PROTEIN TARGETING TO STARCH2b (PTST2b) and MYOSIN RESEMBLING CHLOROPLAST PROTEIN (MRC) on tuber starch granule morphology. A gene duplication event in the Solanaceae resulted in two PTST2 paralogs (PTST2a and PTST2b). PTST2b is expressed in potato tubers, and unlike PTST2a, it had no detectable interaction with STARCH SYNTHASE 4. MRC expression was detectable in leaves, but not in tubers. Using transgenic potato lines in the variety Clearwater Russet, we demonstrate that MRC overexpression leads to the formation of granules with aberrant shapes, many of which arise from multiple initiation points. Silencing PTST2b led to the production of striking near-spherical granules, each arising from a single, central initiation point. Contrary to all reported PTST2 mutants in other species, we observed no change in the number of granules per cell in these lines, suggesting PTST2b is specifically involved in the control of starch granule shape. Starch content and tuber yield per plant were not affected by PTST2b silencing, but MRC overexpression led to strong decreases in both parameters. Notably, the spherical granules in PTST2b silencing lines had a distinctively altered pasting profile, with higher peak and final viscosity than the wild type. Thus, PTST2b and MRC are promising target genes for altering starch granule size and shape in potato tubers, and can be used to create novel starches with altered physicochemical and/or functional properties.
{"title":"Distinct effects of PTST2b and MRC on starch granule morphogenesis in potato tubers","authors":"Anton Hochmuth, Matthew Carswell, Aaron Rowland, Danielle Scarbrough, Lara Esch, Nitin Uttam Kamble, Jeffrey W. Habig, David Seung","doi":"10.1111/pbi.14505","DOIUrl":"10.1111/pbi.14505","url":null,"abstract":"<p>The molecular mechanisms underpinning the formation of the large, ellipsoidal starch granules of potato tuber are poorly understood. Here, we demonstrate the distinct effects of PROTEIN TARGETING TO STARCH2b (PTST2b) and MYOSIN RESEMBLING CHLOROPLAST PROTEIN (MRC) on tuber starch granule morphology. A gene duplication event in the <i>Solanaceae</i> resulted in two PTST2 paralogs (PTST2a and PTST2b). PTST2b is expressed in potato tubers, and unlike PTST2a, it had no detectable interaction with STARCH SYNTHASE 4. MRC expression was detectable in leaves, but not in tubers. Using transgenic potato lines in the variety Clearwater Russet, we demonstrate that MRC overexpression leads to the formation of granules with aberrant shapes, many of which arise from multiple initiation points. Silencing PTST2b led to the production of striking near-spherical granules, each arising from a single, central initiation point. Contrary to all reported PTST2 mutants in other species, we observed no change in the number of granules per cell in these lines, suggesting PTST2b is specifically involved in the control of starch granule shape. Starch content and tuber yield per plant were not affected by PTST2b silencing, but MRC overexpression led to strong decreases in both parameters. Notably, the spherical granules in PTST2b silencing lines had a distinctively altered pasting profile, with higher peak and final viscosity than the wild type. Thus, PTST2b and MRC are promising target genes for altering starch granule size and shape in potato tubers, and can be used to create novel starches with altered physicochemical and/or functional properties.</p>","PeriodicalId":221,"journal":{"name":"Plant Biotechnology Journal","volume":"23 2","pages":"412-429"},"PeriodicalIF":10.1,"publicationDate":"2024-12-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1111/pbi.14505","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142804520","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Achen Zhao, Qiuyi Li, Pengfei Meng, Ping Liu, Siqun Wu, Zhaobo Lang, Yi Song, Alberto P. Macho
Bacteria within the Ralstonia solanacearum species complex cause devastating diseases in numerous crops, causing important losses in food production and industrial supply. Despite extensive efforts to enhance plant tolerance to disease caused by Ralstonia, efficient and sustainable approaches are still missing. Before, we found that Ralstonia promotes the production of gamma-aminobutyric acid (GABA) in plant cells; GABA can be used as a nutrient by Ralstonia to sustain the massive bacterial replication during plant colonization. In this work, we used CRISPR-Cas9-mediated genome editing to mutate SlGAD2, which encodes the major glutamate decarboxylase responsible for GABA production in tomato, a major crop affected by Ralstonia. The resulting Slgad2 mutant plants show reduced GABA content, and enhanced tolerance to bacterial wilt disease upon Ralstonia inoculation. Slgad2 mutant plants did not show altered susceptibility to other tested biotic and abiotic stresses, including drought and heat. Interestingly, Slgad2 mutant plants showed altered microbiome composition in roots and soil. We reveal a strategy to enhance plant resistance to Ralstonia by the manipulation of plant metabolism leading to an impairment of bacterial fitness. This approach could be particularly efficient in combination with other strategies based on the manipulation of the plant immune system, paving the way to a sustainable solution to Ralstonia in agricultural systems.
{"title":"Reduced content of gamma-aminobutyric acid enhances resistance to bacterial wilt disease in tomato","authors":"Achen Zhao, Qiuyi Li, Pengfei Meng, Ping Liu, Siqun Wu, Zhaobo Lang, Yi Song, Alberto P. Macho","doi":"10.1111/pbi.14539","DOIUrl":"https://doi.org/10.1111/pbi.14539","url":null,"abstract":"Bacteria within the <i>Ralstonia solanacearum</i> species complex cause devastating diseases in numerous crops, causing important losses in food production and industrial supply. Despite extensive efforts to enhance plant tolerance to disease caused by <i>Ralstonia</i>, efficient and sustainable approaches are still missing. Before, we found that <i>Ralstonia</i> promotes the production of gamma-aminobutyric acid (GABA) in plant cells; GABA can be used as a nutrient by <i>Ralstonia</i> to sustain the massive bacterial replication during plant colonization. In this work, we used CRISPR-Cas9-mediated genome editing to mutate <i>SlGAD2</i>, which encodes the major glutamate decarboxylase responsible for GABA production in tomato, a major crop affected by <i>Ralstonia</i>. The resulting <i>Slgad2</i> mutant plants show reduced GABA content, and enhanced tolerance to bacterial wilt disease upon <i>Ralstonia</i> inoculation. <i>Slgad2</i> mutant plants did not show altered susceptibility to other tested biotic and abiotic stresses, including drought and heat. Interestingly, <i>Slgad2</i> mutant plants showed altered microbiome composition in roots and soil. We reveal a strategy to enhance plant resistance to <i>Ralstonia</i> by the manipulation of plant metabolism leading to an impairment of bacterial fitness. This approach could be particularly efficient in combination with other strategies based on the manipulation of the plant immune system, paving the way to a sustainable solution to <i>Ralstonia</i> in agricultural systems.","PeriodicalId":221,"journal":{"name":"Plant Biotechnology Journal","volume":"93 1","pages":""},"PeriodicalIF":13.8,"publicationDate":"2024-12-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142793594","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Yitian Liu, Shengrui Zhang, Jing Li, Azam Muhammad, Yue Feng, Jie Qi, Dan Sha, Yushui Hao, Bin Li, Junming Sun
Soybean [Glycine max (L.) Merr.] is an exceptionally rich in isoflavones, and these compounds attach to oestrogen receptors in the human body, lessening the risk of breast cancer and effectively alleviating menopausal syndrome symptoms. Uncovering the molecular mechanisms that regulate soybean isoflavone accumulation is crucial for enhancing the production of these compounds. In this study, we combined bulk segregant analysis sequencing (BSA-seq) and a genome-wide association study (GWAS) to discover a novel R2R3-MYB family gene, GmMYB77, that regulates isoflavone accumulation in soybean. Using the soybean hairy root transient expression system, we verified that GmMYB77 inhibits isoflavone accumulation. Furthermore, knocking out GmMYB77 significantly increased total isoflavone (TIF) content, particularly malonylglycitin, while its overexpression resulted in a notable decrease in contents of malonylglycitin and TIF. We found that GmMYB77 can directly binds the core sequence GGT and suppresses the expression of the key isoflavone biosynthesis genes Isoflavone synthase 1 (GmIFS1), Isoflavone synthase 2 (GmIFS2), Chalcone synthase 7 (GmCHS7) and Chalcone synthase 8 (GmCHS8) by using dual-luciferase assays, electrophoretic mobility shift assays and yeast one-hybrid experiments. Natural variations in the promoter region of GmMYB77 affect its expression, thereby regulating the malonylglycitin and TIF contents. Hap-P2, an elite haplotype, plays a pivotal role in soybean breeding for substantially enhanced isoflavone content. These findings enhance our understanding of the genes influencing soybean isoflavone content and provide a valuable genetic resource for molecular breeding efforts in the future.
{"title":"An R2R3-type MYB transcription factor, GmMYB77, negatively regulates isoflavone accumulation in soybean [Glycine max (L.) Merr.]","authors":"Yitian Liu, Shengrui Zhang, Jing Li, Azam Muhammad, Yue Feng, Jie Qi, Dan Sha, Yushui Hao, Bin Li, Junming Sun","doi":"10.1111/pbi.14541","DOIUrl":"https://doi.org/10.1111/pbi.14541","url":null,"abstract":"Soybean [<i>Glycine max</i> (L.) Merr.] is an exceptionally rich in isoflavones, and these compounds attach to oestrogen receptors in the human body, lessening the risk of breast cancer and effectively alleviating menopausal syndrome symptoms. Uncovering the molecular mechanisms that regulate soybean isoflavone accumulation is crucial for enhancing the production of these compounds. In this study, we combined bulk segregant analysis sequencing (BSA-seq) and a genome-wide association study (GWAS) to discover a novel R2R3-MYB family gene, <i>GmMYB77</i>, that regulates isoflavone accumulation in soybean. Using the soybean hairy root transient expression system, we verified that <i>GmMYB77</i> inhibits isoflavone accumulation. Furthermore, knocking out <i>GmMYB77</i> significantly increased total isoflavone (TIF) content, particularly malonylglycitin, while its overexpression resulted in a notable decrease in contents of malonylglycitin and TIF. We found that GmMYB77 can directly binds the core sequence GGT and suppresses the expression of the key isoflavone biosynthesis genes <i>Isoflavone synthase 1</i> (<i>GmIFS1</i>), <i>Isoflavone synthase 2</i> (<i>GmIFS2</i>), <i>Chalcone synthase 7</i> (<i>GmCHS7</i>) and <i>Chalcone synthase 8</i> (<i>GmCHS8</i>) by using dual-luciferase assays, electrophoretic mobility shift assays and yeast one-hybrid experiments. Natural variations in the promoter region of <i>GmMYB77</i> affect its expression, thereby regulating the malonylglycitin and TIF contents. Hap-P2, an elite haplotype, plays a pivotal role in soybean breeding for substantially enhanced isoflavone content. These findings enhance our understanding of the genes influencing soybean isoflavone content and provide a valuable genetic resource for molecular breeding efforts in the future.","PeriodicalId":221,"journal":{"name":"Plant Biotechnology Journal","volume":"35 1","pages":""},"PeriodicalIF":13.8,"publicationDate":"2024-12-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142789951","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pigments, as coloured secondary metabolites, endow the world with a rich palette of colours. They primarily originate from plants and microorganisms and play crucial roles in their survival and adaptation processes. In this article, we categorize pigments based on their chemical structure into flavonoids, carotenoids, pyrroles, quinones, azaphilones, melanins, betalains, flavins, and others. We further meticulously describe the colours, sources, and biosynthetic pathways, including key enzymatic steps and regulatory networks that control pigment production, in both plants and microorganisms. In particular, we highlight the role of transport proteins and transcription factors in fine-tuning these pathways. Finally, we introduce the use of pigments in practical production and research, aiming to provide new insights and directions for the application of coloured compounds in diverse fields, such as agriculture, industry, and medicine.
{"title":"Natural pigments derived from plants and microorganisms: classification, biosynthesis, and applications","authors":"Qian Tang, Zhibo Li, Ningxin Chen, Xiaozhou Luo, Qiao Zhao","doi":"10.1111/pbi.14522","DOIUrl":"10.1111/pbi.14522","url":null,"abstract":"<p>Pigments, as coloured secondary metabolites, endow the world with a rich palette of colours. They primarily originate from plants and microorganisms and play crucial roles in their survival and adaptation processes. In this article, we categorize pigments based on their chemical structure into flavonoids, carotenoids, pyrroles, quinones, azaphilones, melanins, betalains, flavins, and others. We further meticulously describe the colours, sources, and biosynthetic pathways, including key enzymatic steps and regulatory networks that control pigment production, in both plants and microorganisms. In particular, we highlight the role of transport proteins and transcription factors in fine-tuning these pathways. Finally, we introduce the use of pigments in practical production and research, aiming to provide new insights and directions for the application of coloured compounds in diverse fields, such as agriculture, industry, and medicine.</p>","PeriodicalId":221,"journal":{"name":"Plant Biotechnology Journal","volume":"23 2","pages":"592-614"},"PeriodicalIF":10.1,"publicationDate":"2024-12-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1111/pbi.14522","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142782742","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
<p>Aphids are sap-sucking insects of the order Hemiptera and are considered major agricultural pests owing to their direct feeding damage and transmission of plant viruses (Yu <i>et al</i>., <span>2016</span>). The rapid development of insecticide-resistant pest biotypes and strong dispersal capacity cause significant economic losses in a wide range of plant hosts (Yu <i>et al</i>., <span>2016</span>; Powell <i>et al</i>., <span>2006</span>). Plants expressing <i>Bacillus thuringiensis</i> (Bt) toxins have been successful against lepidopteran and coleopteran pests (Wu <i>et al</i>., <span>2008</span>). However, aphids have evolved into the most abundant pests in Bt crop fields, including in the Bt cotton growing area in China (Lu <i>et al</i>., <span>2010</span>; Yu <i>et al</i>., <span>2016</span>).</p> <p>RNA interference (RNAi) regulates gene expression in a sequence-specific manner in most eukaryotes (Zhao and Guo, <span>2022</span>). In recent years, RNAi-mediated pest control has been achieved via the production of double-stranded RNA (dsRNA) in transgenic plants, a technology referred to as host-induced gene silencing (HIGS), exhibit retarded growth and reduced fecundity or mortality of the corresponding pest species (Dong <i>et al</i>., <span>2024</span>; Mao <i>et al</i>., <span>2011</span>; Zhang <i>et al</i>., <span>2022</span>). However, the effect of RNAi on aphid resistance in cotton plants has not been reported.</p> <p>In this study, to construct the cotton aphid (<i>Aphis gossypii</i>)-specific dsRNA, a gene encoding polyprenyl diphosphate synthase (PDSS) was selected. PDSSs play a critical role in the formation of the prenyl side-chain tail of ubiquinone. Two subunits of aphid long-chain PDSSs designated AgDPPS1 and AgDPPS2, were characterized in <i>Aphis gossypii</i> (Zhang and Li, <span>2013</span>). A 541-bp <i>A. gossypii</i>-specific DPPS1 (KC431243.1) fragment was used to create an RNAi construct for cotton plant transformation (Figure 1a). Southern blot analysis revealed that two individual cotton transformants, AgDPPSi-1 and AgDPPSi-2, each with a single insertion, were obtained (Figure 1b). Small RNA hybridization detected the production of sRNAs in both AgDPPSi lines but not in wild-type (WT) cotton plants (Figure 1c). The offspring of AgDPPSi-1 and AgDPPSi-2, which accumulate sRNAs (Figure S1a), were used for bioassays with cotton aphids. Aphids collected from cotton leaves growing in the natural field were fed on leaves of AgDPPSi and WT cotton plants (Figure S1b). Equal numbers of aphids were fed on the leaves in one plate (Figure 1d). Compared to that at 1 day post-feeding (dpf), the number of total aphids on WT leaves at 3 dpf increased significantly (Figures 1d and S1c). In contrast, the number of total aphids on either AgDPPSi leaf was lower than that at 1 dpf (Figures 1d and S1c). On occasion, aphids moved away quickly, leading to inaccurate numbers of aphids as the initial feeding on the leaves at 1 dpf (Figure
蚜虫是半翅目的吸液昆虫，由于其直接取食破坏和传播植物病毒而被认为是主要的农业害虫（Yu et al., 2016）。抗虫害虫生物型的快速发展和强大的传播能力在广泛的植物寄主中造成重大的经济损失(Yu et al., 2016；Powell et al., 2006)。表达苏云金芽孢杆菌（Bt）毒素的植物已成功对抗鳞翅目和鞘翅目害虫（Wu et al., 2008）。然而，蚜虫已演变为Bt作物田中最常见的害虫，包括中国的Bt棉花种植区(Lu et al., 2010；Yu等人，2016)。RNA干扰（RNAi）在大多数真核生物中以序列特异性的方式调节基因表达（Zhao and Guo, 2022）。近年来，rnai介导的害虫防治已经通过在转基因植物中产生双链RNA （dsRNA）来实现，这种技术被称为宿主诱导基因沉默（HIGS），表现出相应害虫物种的生长迟缓和繁殖能力降低或死亡率降低(Dong et al., 2024；Mao et al., 2011；Zhang等人，2022)。然而，RNAi对棉花蚜虫抗性的影响尚未见报道。为了构建棉蚜（Aphis gossypii）特异性dsRNA，我们选择了一个编码聚戊烯基二磷酸合成酶（PDSS）的基因。PDSSs在泛醌的烯丙基侧链尾部的形成中起关键作用。蚜虫长链PDSSs的两个亚基AgDPPS1和AgDPPS2在棉蚜中被鉴定出来（Zhang and Li, 2013）。一个541 bp的A. gossypii-specific DPPS1 （KC431243.1）片段被用来构建用于棉花植株转化的RNAi构建体（图1a）。Southern blot分析显示，获得了两个单独的棉花转化子，AgDPPSi-1和AgDPPSi-2，每个都有一个插入（图1b）。小RNA杂交在两种AgDPPSi系中检测到sRNAs的产生，但在野生型（WT）棉花中没有检测到（图1c）。AgDPPSi-1和AgDPPSi-2的后代积累了sRNAs（图S1a），用于棉花蚜虫的生物测定。从天然田间生长的棉花叶片中采集蚜虫，以AgDPPSi和WT棉花叶片为食（图S1b）。将等量蚜虫放在一个盘子里喂于叶片上（图1d）。与采食后第1天（dpf）相比，采食后第3天，WT叶片上的蚜虫总数显著增加（图1d和S1c）。相比之下，AgDPPSi叶片上的蚜虫总数低于1 dpf时（图1d和S1c）。有时，蚜虫移动得很快，导致蚜虫的数量不准确，因为在1 dpf时，蚜虫最初以叶子为食（图S1c）。WT叶片上蚜虫数量的增加可能与取食蚜虫的繁殖有关，但也可能与取食AgDPPSi叶片的蚜虫繁殖有关。然而，在3 dpf时，AgDPPSi叶片上观察到少量蚜虫死亡（图1d和S1c）。更准确地说，我们把叶子分别放在盘子里重复生物测定。取食WT叶片后，3 dpf时蚜虫总数显著增加，若虫数量多于1 dpf时（图1e和S1d）。在进食若虫较少的AgDPPSi叶片时，观察到存活蚜虫数量减少，同时死亡蚜虫数量增加（图1e和S1d）。我们拍摄了72小时的延时图像来记录蚜虫在WT和AgDPPSi-2叶片上的实际表现（图S2a）。每片叶子被喂食40只蚜虫并开始射击，视频被缩短到大约100秒。第1天，蚜虫在叶片上的活动非常活跃（图S2a， 0-33秒），第2天蚜虫活动减弱（图S2a， ~ 33-66秒）。第1天在WT叶片上首次观察到新生若虫，第3天数量增加（图S2b，红色圆圈）。而在AgDPPSi-2叶片上则很少观察到若虫。此外，蚜虫看起来很可能没有生命，最终会死在AgDPPSi-2叶片上（图S2a，b）。这些结果表明，AgDPPSi植物有效地降低了蚜虫的存活率和繁殖力。对WT和AgDPPSi-1的5个单叶蚜虫进行计数。结果证实，与1 dpf相比，以3 dpf的WT叶片为食的蚜虫数量有所增加（图1f），但以AgDPPSi-1的叶片为食的蚜虫数量有所减少（图1f），表明AgDPPSi降低了蚜虫的存活率和繁殖力，并导致蚜虫显著死亡（约50%）。以AgDPPSi-2叶片为食的蚜虫死亡率相似。然后，我们通过RT-qPCR分析了从WT和AgDPPSi叶片中收集的蚜虫中AgDPPS1基因的表达水平。与生物测定结果一致，与喂食WT叶片的蚜虫相比，喂食AgDPPSi-1或AgDPPSi-2叶片的蚜虫AgDPPS1基因的表达水平显著降低（图1g）。 图1在图视图中打开ppt抗棉蚜转基因棉花品系的开发。(a) RNAi构建物35S-AgDPPS1i示意图。(b)转基因棉花植株的Southern blot检测。检测到两个转基因棉花品系AgDPPS-1和-2，每个品系都有一个单插入。(c) agdppsi衍生sRNAs的Northern blot检测。（d, e）棉蚜对WT和AgDPPSi-1棉花叶片的取食试验。(f) WT和AgDPPSi-1叶片蚜虫数量变化。(g)采食叶片3 dpf时蚜虫AgDPPS1基因的相对表达。(h)测定天然棉花种植区AgDPPSi-2型棉花植株的抗蚜性。展示了棉花工厂的整体视图和几张特写图片，并在整体视图中显示了相应的位置。(i) WT和AgDPPSi-2植株蚜虫数量变化。(j) Northern印迹法检测AgDPPS1的表达。*表示P <； 0.05。接下来，我们在一个天然棉花种植区对AgDPPSi棉花植株的抗蚜性进行了检测。在棉花试验田播种WT和AgDPPSi-2棉花种子。WT和AgDPPSi-2植株之间没有表型差异（图S3a）。整个棉花生长季节均未施用杀虫剂，蚜害依旧严重。然后用网覆盖棉花植株，为蚜虫爆发和行为分析提供一个相对稳定的环境（图S3b）。棉花植株的整体视图如图1所示。拍摄了几张特写照片。放大图片，并标注相应位置（图1h）。总的来说，AgDPPSi-2植株比WT植株更清洁、更健康。在许多WT植物上观察到粘滞和布满灰尘的叶片（图1h）。在WT棉花叶片的叶柄和背面观察到许多成虫和若虫（图1h）。虽然大部分AgDPPSi-2植株干净，几乎没有蚜虫，但在AgDPPSi-2叶片的一些背面也观察到一些蚜虫；然而，它们不像蚜虫感染的WT叶片那样致密和多尘。从WT和AgDPPSi-2植株中随机抽取5株蚜虫感染植株，计数蚜虫数量。2023年7月5日，AgDPPSi-2植株平均蚜虫数（~764只/株）显著低于WT植株（~2034只/株）（图1i）。2023年7月23日，经过多日降雨，蚜虫数量下降（图1i）。AgDPPSi-2植株的平均蚜虫数（约198只/株）低于WT植株（约846只/株）。然后从叶片中收集蚜虫，计数提取蚜虫RNA。从WT叶片和AgDPPSi-2叶片中分离得到混合蚜虫的总rna。Northern blot检测显示，与WT棉花叶片相比，AgDPPSi-2叶片上蚜虫的AgDPPS1 mRNA被降解（图1j），表明AgDPPSi有效地沉默了AgDPPSi-2植株上蚜虫的AgDPPS1 mRNA，从而降低了蚜虫的生存能力。综上所述，我们的数据证明了AgDPPSi棉花在天然棉田中的抗蚜性。综上所述，我们通过表达一种针对棉蚜特异性DPPS1基因的RNAi构建物，开发了转基因棉系。DPPS1沉默会阻碍泛醌的形成。因此，在室内和天
{"title":"HIGS-mediated crop protection against cotton aphids","authors":"Wen Tian, Tao Zhang, Jian-Hua Zhao, Yong-Mei Dong, You-Zhong Li, Zeng-Qiang Zhao, Feng Gao, Xue-Ming Wu, Bo-Sen Zhang, Yuan-Yuan Fang, Zong-Ming Xie, Hui-Shan Guo","doi":"10.1111/pbi.14529","DOIUrl":"https://doi.org/10.1111/pbi.14529","url":null,"abstract":"<p>Aphids are sap-sucking insects of the order Hemiptera and are considered major agricultural pests owing to their direct feeding damage and transmission of plant viruses (Yu <i>et al</i>., <span>2016</span>). The rapid development of insecticide-resistant pest biotypes and strong dispersal capacity cause significant economic losses in a wide range of plant hosts (Yu <i>et al</i>., <span>2016</span>; Powell <i>et al</i>., <span>2006</span>). Plants expressing <i>Bacillus thuringiensis</i> (Bt) toxins have been successful against lepidopteran and coleopteran pests (Wu <i>et al</i>., <span>2008</span>). However, aphids have evolved into the most abundant pests in Bt crop fields, including in the Bt cotton growing area in China (Lu <i>et al</i>., <span>2010</span>; Yu <i>et al</i>., <span>2016</span>).</p>\u0000<p>RNA interference (RNAi) regulates gene expression in a sequence-specific manner in most eukaryotes (Zhao and Guo, <span>2022</span>). In recent years, RNAi-mediated pest control has been achieved via the production of double-stranded RNA (dsRNA) in transgenic plants, a technology referred to as host-induced gene silencing (HIGS), exhibit retarded growth and reduced fecundity or mortality of the corresponding pest species (Dong <i>et al</i>., <span>2024</span>; Mao <i>et al</i>., <span>2011</span>; Zhang <i>et al</i>., <span>2022</span>). However, the effect of RNAi on aphid resistance in cotton plants has not been reported.</p>\u0000<p>In this study, to construct the cotton aphid (<i>Aphis gossypii</i>)-specific dsRNA, a gene encoding polyprenyl diphosphate synthase (PDSS) was selected. PDSSs play a critical role in the formation of the prenyl side-chain tail of ubiquinone. Two subunits of aphid long-chain PDSSs designated AgDPPS1 and AgDPPS2, were characterized in <i>Aphis gossypii</i> (Zhang and Li, <span>2013</span>). A 541-bp <i>A. gossypii</i>-specific DPPS1 (KC431243.1) fragment was used to create an RNAi construct for cotton plant transformation (Figure 1a). Southern blot analysis revealed that two individual cotton transformants, AgDPPSi-1 and AgDPPSi-2, each with a single insertion, were obtained (Figure 1b). Small RNA hybridization detected the production of sRNAs in both AgDPPSi lines but not in wild-type (WT) cotton plants (Figure 1c). The offspring of AgDPPSi-1 and AgDPPSi-2, which accumulate sRNAs (Figure S1a), were used for bioassays with cotton aphids. Aphids collected from cotton leaves growing in the natural field were fed on leaves of AgDPPSi and WT cotton plants (Figure S1b). Equal numbers of aphids were fed on the leaves in one plate (Figure 1d). Compared to that at 1 day post-feeding (dpf), the number of total aphids on WT leaves at 3 dpf increased significantly (Figures 1d and S1c). In contrast, the number of total aphids on either AgDPPSi leaf was lower than that at 1 dpf (Figures 1d and S1c). On occasion, aphids moved away quickly, leading to inaccurate numbers of aphids as the initial feeding on the leaves at 1 dpf (Figure","PeriodicalId":221,"journal":{"name":"Plant Biotechnology Journal","volume":"76 1","pages":""},"PeriodicalIF":13.8,"publicationDate":"2024-12-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142777427","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Mariem Bradai, Huang Tan, Man Gao, Emmanuel Aguilar, Rosa Lozano-Durán
<p>Viruses are obligate intracellular pathogens that manipulate the cells they invade to create an environment conducive to their multiplication and spread. In plants, virus-caused diseases result in severe yield losses. At any given time during infection, only a fraction of cells supports active viral replication, frequently in a tissue-specific manner. It logically follows that, if entire organs (e.g. leaves) are considered when studying virus-induced molecular changes, significant dilution issues and an inability to distinguish cell-autonomous from systemic responses to the infection are faced, which hinder emergence of a clear overview of the impact of the viral invasion. Therefore, methods to specifically distinguish infected cells are required in plant virus research. While some viruses have been engineered to contain a reporter gene in their genomes, for example, encoding a fluorescent protein, to enable monitoring of the infection, these changes are frequently not possible and/or affect the dynamics of the infection.</p> <p>Geminiviruses belong to a family of plant-infecting viruses with circular, single-stranded (ss) DNA genomes and causal agents of devastating diseases in crops worldwide. Geminiviruses replicate in the nucleus of the infected cell through a combination of rolling-circle replication (RCR) and recombination-dependent replication (RDR), utilising the plant DNA replication machinery and only one viral protein, Rep (for replication-associated). Rep recognises the origin of replication present in the intergenic region (IR) of the viral genome, recruits the necessary plant factors, and introduces a nick in the complementary strand of the double-stranded replicative intermediate; RCR ensues, generating multiple copies of the viral genome, with contribution of RDR (Hanley-Bowdoin <i>et al</i>., <span>2013</span>).</p> <p>To monitor the dynamics of the geminiviral infection in space and time with cellular resolution, a simple, visual, and non-destructive method is needed. RCR can be co-opted to enable the Rep-dependent replication of extra-chromosomal replicons (ECRs) containing sequences of interest. In this approach, the sequence of choice is flanked by two direct repeats of the viral IR; when Rep is provided <i>in trans</i>, through transgenic expression or during infection, the replication of this sequence as an ECR leads to the accumulation of the encoded protein to very high levels (Figure 1a; Morilla <i>et al</i>., <span>2006</span>). Transgenic reporter plants containing this type of construct have been successfully employed to monitor the geminiviral infection and to evaluate virulence in reverse-genetic experiments in model plants (Kato <i>et al</i>., <span>2020</span>; Krenz <i>et al</i>., <span>2015</span>; Lozano-Durán <i>et al</i>., <span>2011</span>; Morilla <i>et al</i>., <span>2006</span>). However, these approaches are still lacking in crop species.</p> <figure><picture> <source media="(min-width: 1650px)" srcse
病毒是专性的细胞内病原体，操纵它们入侵的细胞，创造有利于它们繁殖和传播的环境。在植物中，病毒引起的疾病导致严重的产量损失。在感染期间的任何给定时间，只有一小部分细胞支持活跃的病毒复制，通常以组织特异性的方式进行。从逻辑上讲，如果在研究病毒诱导的分子变化时考虑整个器官（例如叶子），就会面临严重的稀释问题以及无法区分细胞自主反应和对感染的全身反应，这阻碍了对病毒入侵影响的清晰概述的出现。因此，在植物病毒研究中，需要找到特异性区分感染细胞的方法。虽然一些病毒已被改造成在其基因组中含有报告基因，例如编码荧光蛋白，以便监测感染，但这些变化通常是不可能的，并且/或影响感染的动态。双病毒属于一个具有环状单链DNA基因组的植物感染病毒家族，是全世界农作物毁灭性疾病的致病因子。双病毒通过滚环复制（RCR）和重组依赖复制（RDR）的组合在受感染细胞的细胞核中复制，利用植物DNA复制机制和只有一种病毒蛋白，Rep（复制相关）。Rep识别存在于病毒基因组基因间区（IR）的复制起源，招募必要的植物因子，并在双链复制中间体的互补链中引入缺口；RCR随后发生，在RDR的作用下产生病毒基因组的多个拷贝（Hanley-Bowdoin et al., 2013）。为了以细胞分辨率监测双病毒感染在空间和时间上的动态，需要一种简单、直观和非破坏性的方法。RCR可以被增选，使含有感兴趣序列的染色体外复制子（ecr）的rep依赖复制成为可能。在这种方法中，选择的序列两侧是病毒IR的两个直接重复序列；当Rep以反式、通过转基因表达或在感染期间提供时，该序列作为ECR的复制导致编码蛋白的积累达到非常高的水平(图1a；Morilla et al., 2006)。含有这种构建体的转基因报告植物已成功用于监测双病毒感染，并在模式植物的反向遗传实验中评估毒力(Kato等人，2020；Krenz et al., 2015；Lozano-Durán等人，2011；Morilla et al., 2006)。然而，这些方法在作物品种中仍然缺乏。图1打开图形查看器powerpoint2ir - dsred转基因番茄植株的生成和表征。(a) 2IR-DsRed结构。IR: TYLCV基因间区；35S pro: 35S花椰菜花叶病毒启动子；NosT: NOS终止符。在TYLCV感染后，病毒Rep特异性结合IR，触发DsRed染色体外复制子（ecr）的产生，导致DsRed转基因的过表达和荧光蛋白的积累。在没有TYLCV（“非感染植物”）的情况下，基底的DsRed积累量很低。(b)水培培养的tylcv感染2IR-DsRed植株的症状。分别于接种后9、16、23天拍照。比例尺= 10厘米。在荧光立体显微镜下，在没有（模拟）和存在（TYLCV）病毒的代表性器官中观察到DsRed信号。根和叶的图像如(c)所示；手切叶柄和茎的图像见(d)。比例尺= 1毫米。(e)共聚焦显微镜下叶柄和茎段的代表性照片。在14 dpi时取样。箭头表示韧皮部细胞支持病毒复制。标尺= 200 μm。通过两步锚定定量PCR （qPCR）测量病毒链（VS） （f, g）和互补链（CS） （h, i）的积累（Rodríguez-Negrete et al., 2014）；DsRed (j, k)， qPCR定量。数值呈现相对于25S核糖体DNA间隔（ITS）。在9、16和23 dpi时，分别从两株未感染植株（模拟P1和P2）和两株独立2IR-DsRed品系（TYLCV、P1、P2和P3）的最顶端叶片和三株感染植株上采集样本。采用Wu et al.（2021）的方法提取DNA并进行qPCR。在（b-k）中进行病毒感染试验时，将携带TYLCV感染克隆（OD600 = 0.1）的农杆菌注射接种于2 ~ 3周龄植株的茎中。引物列表见表S1。(l) 2IR-DsRed植物中DsRed信号的进展。三个层次的表达，高（红色），低（粉红色），和无(绿色，叶子；灰色表示根。数字表示叶片相对于接种点的位置。 为了方便实时跟踪病毒在受双病毒引起的疾病严重影响的作物体内的入侵情况，我们培育了转基因报告基因番茄茄(Solanum lycopersicum，番茄，cv。Moneymaker)植物含有一个结构体，该结构体包括侵染番茄的番茄黄卷叶病毒（TYLCV）的两个直接重复IR，在盒式两侧表达盘状虫红色荧光蛋白（DsRed）（图1a），命名为2IR-DsRed（在p<s:1> rez- padilla et al.， 2020中描述）。在感染期间，TYLCV Rep蛋白特异性识别盒侧的IRs并触发ecr的产生，导致荧光蛋白的高积累（图1a）。因此，诱导DsRed积累与Rep活性直接相关，从而与病毒复制相关，从而可以以简单直观的方式监测整个植物的感染发展。接下来，我们利用这些植物利用DsRed积累作为病毒复制的代理来跟踪TYLCV感染。两个独立的转基因品系在水培条件下生长，并在萌发后2周与一个TYLCV感染克隆进行农接种，并在接种后9、16和23天（dpi）对地上部分和根部的DsRed荧光进行监测。在受感染的植物中观察到典型的生长减少（图1b）。在tylcv感染植株的叶、茎、叶柄和根的脉管系统中检测到强烈的DsRed荧光（图1c，d），而模拟接种植株的背景荧光消失；图11总结了指示病毒复制的DsRed荧光的进展情况。值得注意的是，在所有三次采样中，都可以在地上部分和根部观察到活跃的病毒复制，在2到3周之间达到峰值(图1c；图S1)。在共聚焦显微镜下观察叶柄横切面和茎横切面和纵切面显示，单个韧皮部细胞支持病毒复制(图1e；图S2)。重要的是，视觉DsRed荧光与病毒DNA积累（病毒链VS和互补链CS，仅存在于复制中间体中）以及含有DsRed的ecr的积累相关（图1f-k）。因此，2IR-DsRed番茄植株是实时跟踪TYLCV在其天然宿主番茄中的复制的可靠工具，具有非破坏性和成本效益，并且具有细胞分辨率，可用于评估不同因素（非生物或生物）如何在中通量或高通量方法中影响病毒感染。此外，荧光标记的存在将使通过荧光辅助细胞分选（FACS）或激光辅助显微解剖分离支持活性复制的受感染细胞进行分子分析成为可能。病毒诱导基因沉默（VIGS）是一种适用于番茄植物的反向遗传学方法，也可以与2IR-DsRed报告植物结合使用，以简单、中等通量和廉价的方式鉴定病毒入侵所需的植物基因，如之前在烟叶上的类似系统所证明的那样（Lozano-Durán等人，2011）。考虑到本文的结果，我们认为2IR-DsRed植株提供了一种经济实惠的系统，可以快速、简单地监测番茄TYLCV感染，从而筛选影响该过程的因素并鉴定和分离感染细胞，因此代表了植物病毒研究的有价值的工具。这些细胞系的特征也揭示了TYLCV在感染期间在不同器官中活跃复制的动力学，并揭示了番茄根部持续的病毒复制。
{"title":"A reporter tomato line to track replication of a geminivirus in real time and with cellular resolution","authors":"Mariem Bradai, Huang Tan, Man Gao, Emmanuel Aguilar, Rosa Lozano-Durán","doi":"10.1111/pbi.14531","DOIUrl":"https://doi.org/10.1111/pbi.14531","url":null,"abstract":"<p>Viruses are obligate intracellular pathogens that manipulate the cells they invade to create an environment conducive to their multiplication and spread. In plants, virus-caused diseases result in severe yield losses. At any given time during infection, only a fraction of cells supports active viral replication, frequently in a tissue-specific manner. It logically follows that, if entire organs (e.g. leaves) are considered when studying virus-induced molecular changes, significant dilution issues and an inability to distinguish cell-autonomous from systemic responses to the infection are faced, which hinder emergence of a clear overview of the impact of the viral invasion. Therefore, methods to specifically distinguish infected cells are required in plant virus research. While some viruses have been engineered to contain a reporter gene in their genomes, for example, encoding a fluorescent protein, to enable monitoring of the infection, these changes are frequently not possible and/or affect the dynamics of the infection.</p>\u0000<p>Geminiviruses belong to a family of plant-infecting viruses with circular, single-stranded (ss) DNA genomes and causal agents of devastating diseases in crops worldwide. Geminiviruses replicate in the nucleus of the infected cell through a combination of rolling-circle replication (RCR) and recombination-dependent replication (RDR), utilising the plant DNA replication machinery and only one viral protein, Rep (for replication-associated). Rep recognises the origin of replication present in the intergenic region (IR) of the viral genome, recruits the necessary plant factors, and introduces a nick in the complementary strand of the double-stranded replicative intermediate; RCR ensues, generating multiple copies of the viral genome, with contribution of RDR (Hanley-Bowdoin <i>et al</i>., <span>2013</span>).</p>\u0000<p>To monitor the dynamics of the geminiviral infection in space and time with cellular resolution, a simple, visual, and non-destructive method is needed. RCR can be co-opted to enable the Rep-dependent replication of extra-chromosomal replicons (ECRs) containing sequences of interest. In this approach, the sequence of choice is flanked by two direct repeats of the viral IR; when Rep is provided <i>in trans</i>, through transgenic expression or during infection, the replication of this sequence as an ECR leads to the accumulation of the encoded protein to very high levels (Figure 1a; Morilla <i>et al</i>., <span>2006</span>). Transgenic reporter plants containing this type of construct have been successfully employed to monitor the geminiviral infection and to evaluate virulence in reverse-genetic experiments in model plants (Kato <i>et al</i>., <span>2020</span>; Krenz <i>et al</i>., <span>2015</span>; Lozano-Durán <i>et al</i>., <span>2011</span>; Morilla <i>et al</i>., <span>2006</span>). However, these approaches are still lacking in crop species.</p>\u0000<figure><picture>\u0000<source media=\"(min-width: 1650px)\" srcse","PeriodicalId":221,"journal":{"name":"Plant Biotechnology Journal","volume":"37 1","pages":""},"PeriodicalIF":13.8,"publicationDate":"2024-12-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142777431","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
<p>Trees account for approximately 90% of the Earth's biomass and provide humans with various necessities for survival, such as clean air and water, wood, fibre and fuel (Petit and Hampe, <span>2006</span>; Tuskan <i>et al</i>., <span>2006</span>). Compared with annual plants, trees have many significant features, such as perennial growth, large size, secondary growth from a vascular cambium and dormancy (Douglas, <span>2017</span>). Trees should be considered as a model system in plant biology and provide possibilities to answer questions that cannot be easily solved in the annual model systems of <i>Arabidopsis</i> and rice.</p> <p>Trees of the genus <i>Populus</i> are prominent forest species in temperate regions of the Northern Hemisphere. <i>Populus</i> trees, as the model systems for plant biology, have several advantages, including rapid growth, small genome, facile transgenesis and easy cloning (Bradshaw <i>et al</i>., <span>2000</span>). <i>Populus trichocarpa</i> is now widely used as a model system in the United States and Europe. <i>Populus trichocarpa</i> native to western North American cannot grow well in the fields and forests of China, which limits its breeding and application potential in China. In China, <i>Populus tomentosa</i> used to be a study system in basic research. It is a stabilized interspecific hybrid species widespread in Asia and is extensively used in breeding or forestry industries. Besides, poplar 84 K (<i>Populus alba</i> × <i>P. glandulosa</i>) and poplar 741 (<i>Populus alba</i> × (<i>P. davidiana</i> × <i>P. simonii</i>) × <i>P. tomentosa</i>) are also widely used as study systems in China. These two cultivars and <i>P. tomentosa</i> are not the most suitable strains for tree genetic study system due to their hybridization background.</p> <p>The white poplar (<i>P. alba</i>) is a widespread forest tree in the world, distributed in Europe, Asia, North America, South America, Africa and Oceania (Figure 1a). <i>Populus alba</i> is not only a beautiful tree (Figure 1b) but also has extensive adaptability to different ecological environments (Stölting <i>et al</i>., <span>2015</span>). As a fast growing tree, <i>P. alba</i> is included in the forest tree breeding programme in China and the European programme of forest genetic resources. Our previous study found that with the natural expansion of <i>P. alba</i> from Europe to China, the natural populations in China experienced a bottleneck effect. Average pooled heterozygosity value of <i>P. alba</i> populations in China was much lower than that in Italy and Hungary (Liu <i>et al</i>., <span>2019</span>). As a pure and highly adaptable natural species, <i>P. alba</i> has been used as a hybrid parent in the breeding history of China. Due to the widespread distribution of <i>P. alba</i> in the Eurasian continent, using <i>P. alba</i> as a research system is conducive to the promotion of research results. Thus, <i>P. alba</i> in China is suitable as a study system fo
树木约占地球生物量的90%，为人类提供各种生存必需品，如清洁的空气和水、木材、纤维和燃料(Petit和Hampe, 2006；Tuskan et al., 2006)。与一年生植物相比，乔木具有多年生、体积大、维管形成层次生和休眠等显著特征（Douglas, 2017）。在植物生物学中，树木应该被视为一种模式系统，并为回答拟南芥和水稻的年度模式系统中不易解决的问题提供了可能性。杨树属树木是北半球温带地区重要的森林树种。杨树作为植物生物学的模式系统，具有生长快、基因组小、易转基因、易克隆等优点（Bradshaw et al., 2000）。在美国和欧洲，毛杨作为一种模式系统被广泛使用。原产于北美西部的毛卡杨在中国的田间和森林生长不佳，限制了其在中国的育种和应用潜力。在中国，毛白杨曾是基础研究中的一个研究体系。它是一种稳定的种间杂交种，广泛分布于亚洲，广泛用于育种或林业。此外，84 K（白杨×甘绿杨）和741 K（白杨×大叶杨）×甘绿杨。毛毡)也被广泛用作中国的学习系统。由于其杂交背景，这两个品种和毛毛白杨都不是树遗传研究系统的最合适品系。白杨树（P. alba）是世界上广泛分布的森林乔木，分布在欧洲、亚洲、北美、南美、非洲和大洋洲（图1a）。白杨不仅是一棵美丽的树（图1b），而且对不同的生态环境具有广泛的适应性（Stölting et al., 2015）。白杨是一种生长迅速的乔木，已被列入中国林木育种计划和欧洲森林遗传资源计划。我们的前期研究发现，随着白杨从欧洲向中国的自然扩张，中国的自然种群经历了瓶颈效应。中国白杨种群的平均汇总杂合度远低于意大利和匈牙利（Liu et al., 2019）。作为一种纯种、适应性强的天然种，白藻在中国育种史上一直被用作杂交亲本。由于白藻在欧亚大陆分布广泛，将白藻作为研究体系有利于研究成果的推广。因此，中国白杨适合作为树木生物学的研究体系。图1在图视图中打开powerpointpopulus alba v2.0基因组特征和CRISPR/Cas9系统（详见文本S1）。白杨v1.0组装片段化，由1285个contigs组成，N50为1181 Kb，并补充了两个细胞器基因组片段（Liu et al, 2019）。在这里，我们提出了一个基于染色体构象捕获的新组装，同时结合了以前的太平洋生物科学（PacBio）单分子实时（SMRT）和全基因组霰弹枪序列。共获得124.17 Gb的Illumina clean数据用于高通量染色体构象捕获（Hi-C）分析（表S1）。基于这些数据，将P. alba的contigs聚类成97个支架，最终N50为22.7 Mb（表S2）。这97个支架包括19个染色体长度的假分子，覆盖412.7 Mb的基因组序列和78个未放置的支架，代表3.7 Mb的未整合序列。根据假分子与P. trichocarpa v4基因组的共线性，对19个染色体长度的假分子进行排序（图1c）。在P. alba v1.0组装中，所有被认为是线粒体和质体来源的序列在搭建之前被移除，并分别组装成完整的叶绿体和线粒体基因组（图S1和S2）。检测Benchmarking Universal Single-Copy Orthologs （BUSCO）、Core Eukaryotic gene （CEGMA）和LTR Assembly Index （LAI）的预期基因含量，验证基因组组装的完整性（表S3）。结果显示，在P. alba v2.0基因组中可以检测到97.8%的真核生物、98.4%的胚胎植物和98.4%的真核生物同源物（图1d）。BUSCO、CEGMA和LAI的评价结果均有不同程度的提高，表明P. alba v2.0基因组的连续性和完整性较v1.0有所提高。P. alba v2.0基因组的BUSCO和CEGMA完整性与P. trichocarpa v4接近。LAI评分大于10，表明该群落已达到森林参考质量水平。根据亲缘物种的表达数据和基因集，通过优化注释程序，在注释2.0版本中获得34 010个预测蛋白编码基因模型。 其中29 847例（87.76%）至少有一个数据库进行了功能标注（图1e）。在所有的基因模型中，有21 143个（62.16%）可以发现片段重复衍生的同源物（图1f）。在P. alba v2.0中发现的重复元素略多于v1.0（表S4）。绝大多数te为LTR-RT，以gypsy型元素为主，占所有重复元素的45.3%，其次是copia型，占17.9%。对拟南芥和毛卡藻的研究表明，着丝粒区域基因稀疏，SNP密度低，吉普赛元素富集，胞嘧啶甲基化程度高(Jiang et al., 2003；Natali et al., 2015；Weighill et al., 2019)。中心点位置预测使用了由Weighill等人（2019）描述的基于小波的基因组信号分析(图1g-i；图S3)。除2、7、8、15、18和19条假染色体外，其余13条假染色体的相对着丝粒位置(中心bin （bp)/染色体长度（bp））与毛藻假染色体相似(表S5；图S4)。通过将毛霉和各种植物的着丝粒重复序列定位到v2.0染色体上，可以进一步确定着丝粒/着丝粒在染色体1、3、6、9、11、12、18和19上的位置（图S3）。在标准农杆菌介导转化方法的基础上，我们优化了已测序的白杨植物的转化程序（补充材料和方法）。外植体最初从灭菌的嫩茎中获得，然后每4周进行继代培养以持续利用。报告基因、β-葡萄糖醛酸酶（GUS）和P. alba植物烯去饱和酶（PDS）基因分别用于检测转化和基因组编辑效率。利用组培苗的叶片和茎段进行转化。玉米素（ZT）是一种促进非分生组织分裂的有效植物激素。为了提高转化效率，研究了在添加2 ~ 100 mg/L ZT的培养基中培养的叶片和茎段愈伤组织再生率。结果表明，相对低浓度的ZT （2 ~ 10 mg/L）足以使愈伤组织在4周内再生（图1j）。叶片和茎段ZT的最佳浓度不同。根据该方案，在与农杆菌共培养后4 ~ 5周和10 ~ 12周内，分别成功诱导芽和根再生。最终转化效率为31.96%。PDS的基因组编辑效率为69.47%（图1k， 1）。这种高效的遗传转化方法将有助于白藻的功能研究。
{"title":"Genome and CRISPR/Cas9 system of a widespread forest tree (Populus alba) in the world","authors":"Yan-Jing Liu, Peng-Fei Jiang, Xue-Min Han, Xiao-Yuan Li, Hai-Ming Wang, Yun-Jiao Wang, Xiao-Xia Wang, Qing-Yin Zeng","doi":"10.1111/pbi.14543","DOIUrl":"https://doi.org/10.1111/pbi.14543","url":null,"abstract":"<p>Trees account for approximately 90% of the Earth's biomass and provide humans with various necessities for survival, such as clean air and water, wood, fibre and fuel (Petit and Hampe, <span>2006</span>; Tuskan <i>et al</i>., <span>2006</span>). Compared with annual plants, trees have many significant features, such as perennial growth, large size, secondary growth from a vascular cambium and dormancy (Douglas, <span>2017</span>). Trees should be considered as a model system in plant biology and provide possibilities to answer questions that cannot be easily solved in the annual model systems of <i>Arabidopsis</i> and rice.</p>\u0000<p>Trees of the genus <i>Populus</i> are prominent forest species in temperate regions of the Northern Hemisphere. <i>Populus</i> trees, as the model systems for plant biology, have several advantages, including rapid growth, small genome, facile transgenesis and easy cloning (Bradshaw <i>et al</i>., <span>2000</span>). <i>Populus trichocarpa</i> is now widely used as a model system in the United States and Europe. <i>Populus trichocarpa</i> native to western North American cannot grow well in the fields and forests of China, which limits its breeding and application potential in China. In China, <i>Populus tomentosa</i> used to be a study system in basic research. It is a stabilized interspecific hybrid species widespread in Asia and is extensively used in breeding or forestry industries. Besides, poplar 84 K (<i>Populus alba</i> × <i>P. glandulosa</i>) and poplar 741 (<i>Populus alba</i> × (<i>P. davidiana</i> × <i>P. simonii</i>) × <i>P. tomentosa</i>) are also widely used as study systems in China. These two cultivars and <i>P. tomentosa</i> are not the most suitable strains for tree genetic study system due to their hybridization background.</p>\u0000<p>The white poplar (<i>P. alba</i>) is a widespread forest tree in the world, distributed in Europe, Asia, North America, South America, Africa and Oceania (Figure 1a). <i>Populus alba</i> is not only a beautiful tree (Figure 1b) but also has extensive adaptability to different ecological environments (Stölting <i>et al</i>., <span>2015</span>). As a fast growing tree, <i>P. alba</i> is included in the forest tree breeding programme in China and the European programme of forest genetic resources. Our previous study found that with the natural expansion of <i>P. alba</i> from Europe to China, the natural populations in China experienced a bottleneck effect. Average pooled heterozygosity value of <i>P. alba</i> populations in China was much lower than that in Italy and Hungary (Liu <i>et al</i>., <span>2019</span>). As a pure and highly adaptable natural species, <i>P. alba</i> has been used as a hybrid parent in the breeding history of China. Due to the widespread distribution of <i>P. alba</i> in the Eurasian continent, using <i>P. alba</i> as a research system is conducive to the promotion of research results. Thus, <i>P. alba</i> in China is suitable as a study system fo","PeriodicalId":221,"journal":{"name":"Plant Biotechnology Journal","volume":"137 1","pages":""},"PeriodicalIF":13.8,"publicationDate":"2024-12-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142777444","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Salinity significantly inhibits plant growth and development. While the recretohalophyte Limonium bicolor can reduce its ion content by secreting salt, the metabolic pathways it employs to adapt to high salt stress remain unclear. This study aims to unravel this enigma through integrated transcriptomic and metabolomic analyses of L. bicolor under salt stress conditions. The results showed that compared to the control (S0), low salt treatment (S1) led to a significant increase in plant growth, photosynthesis efficiency and antioxidant enzyme activity but caused no significant changes in organic soluble substance and ROS contents. However, high salt treatments (S3 and S4) led to a significant decrease in plant growth, photosynthesis efficiency and antioxidant enzyme activity, accompanied by a significant increase in organic soluble substance and ROS contents. A significant increase in phenolic compounds, such as caffeoyl shikimic acid and coniferin, upon the treatments of S1, S3 and S4, and a decrease and increase in flavonoids upon the treatments of S1 and S3 were also observed, respectively. This study also demonstrated that the expression patterns of key genes responsible for the biosynthesis of these metabolites are consistent with the observed trends in their accumulation levels. These results suggest that under low salt stress conditions, the halophyte L. bicolor experiences minimal osmotic and oxidative stress. However, under high salt stress conditions, it suffers severe osmotic and oxidative stress, and the increase in organic soluble substances and flavonoids serves as a key response to these stresses and also represents a good strategy for the alleviation of them.
{"title":"Integrated transcriptomic and metabolomic analyses uncover the key pathways of Limonium bicolor in response to salt stress","authors":"Zhihui Zhu, Yuqing Zhou, Xiuyue Liu, Fanxia Meng, Chenhan Xu, Min Chen","doi":"10.1111/pbi.14534","DOIUrl":"https://doi.org/10.1111/pbi.14534","url":null,"abstract":"Salinity significantly inhibits plant growth and development. While the recretohalophyte <i>Limonium bicolor</i> can reduce its ion content by secreting salt, the metabolic pathways it employs to adapt to high salt stress remain unclear. This study aims to unravel this enigma through integrated transcriptomic and metabolomic analyses of <i>L. bicolor</i> under salt stress conditions. The results showed that compared to the control (S0), low salt treatment (S1) led to a significant increase in plant growth, photosynthesis efficiency and antioxidant enzyme activity but caused no significant changes in organic soluble substance and ROS contents. However, high salt treatments (S3 and S4) led to a significant decrease in plant growth, photosynthesis efficiency and antioxidant enzyme activity, accompanied by a significant increase in organic soluble substance and ROS contents. A significant increase in phenolic compounds, such as caffeoyl shikimic acid and coniferin, upon the treatments of S1, S3 and S4, and a decrease and increase in flavonoids upon the treatments of S1 and S3 were also observed, respectively. This study also demonstrated that the expression patterns of key genes responsible for the biosynthesis of these metabolites are consistent with the observed trends in their accumulation levels. These results suggest that under low salt stress conditions, the halophyte <i>L. bicolor</i> experiences minimal osmotic and oxidative stress. However, under high salt stress conditions, it suffers severe osmotic and oxidative stress, and the increase in organic soluble substances and flavonoids serves as a key response to these stresses and also represents a good strategy for the alleviation of them.","PeriodicalId":221,"journal":{"name":"Plant Biotechnology Journal","volume":"9 1","pages":""},"PeriodicalIF":13.8,"publicationDate":"2024-12-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142777430","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Sheath blight (ShB), caused by Rhizoctonia solani, is a highly destructive disease in many crops worldwide and no major resistance genes are available. Here, we identified a sbr1 (sheath blight resistance 1) rice mutant, which shows enhanced ShB resistance and maintains wildtype agronomic traits including yield, but carries an undesired stay-green phenotype. Through map-based cloning and transgenic validation, we found that an insertion disrupting the Stay-Green (OsSGR) gene is responsible for sbr1 phenotypes. Mechanistically, the sbr1/Ossgr mutants reduce the expression of most OsCKX genes, which function in cytokinin (CK) degradation, to accumulate CK leading to ShB resistance. Importantly, knockout of OsCKX7, predominantly expressed in the leaf sheath and highly induced by R. solani, significantly enhances ShB resistance without stay-green phenotype nor yield penalty, showing high application potential. Thus, our study reveals novel insights that OsSGR and cytokinin play key roles in rice-R. solani interaction and generates a valuable ShB-resistant germplasm.
{"title":"Natural mutation in Stay-Green (OsSGR) confers enhanced resistance to rice sheath blight through elevating cytokinin content","authors":"Wenya Xie, Xiang Xue, Yu Wang, Guiyun Zhang, Jianhua Zhao, Huimin Zhang, Guangda Wang, Lei Li, Yiqin Wang, Wenfeng Shan, Yafang Zhang, Zongxiang Chen, Xijun Chen, Zhiming Feng, Keming Hu, Mingfa Sun, Chengcai Chu, Shimin Zuo","doi":"10.1111/pbi.14540","DOIUrl":"https://doi.org/10.1111/pbi.14540","url":null,"abstract":"Sheath blight (ShB), caused by <i>Rhizoctonia solani</i>, is a highly destructive disease in many crops worldwide and no major resistance genes are available. Here, we identified a <i>sbr1</i> (<i>sheath blight resistance 1</i>) rice mutant, which shows enhanced ShB resistance and maintains wildtype agronomic traits including yield, but carries an undesired stay-green phenotype. Through map-based cloning and transgenic validation, we found that an insertion disrupting the <i>Stay-Green</i> (<i>OsSGR</i>) gene is responsible for <i>sbr1</i> phenotypes. Mechanistically, the <i>sbr1</i>/<i>Ossgr</i> mutants reduce the expression of most <i>OsCKX</i> genes, which function in cytokinin (CK) degradation, to accumulate CK leading to ShB resistance. Importantly, knockout of <i>OsCKX7</i>, predominantly expressed in the leaf sheath and highly induced by <i>R. solani</i>, significantly enhances ShB resistance without stay-green phenotype nor yield penalty, showing high application potential. Thus, our study reveals novel insights that <i>OsSGR</i> and cytokinin play key roles in rice-<i>R. solani</i> interaction and generates a valuable ShB-resistant germplasm.","PeriodicalId":221,"journal":{"name":"Plant Biotechnology Journal","volume":"12 1","pages":""},"PeriodicalIF":13.8,"publicationDate":"2024-12-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142763264","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Yujie Zhou, Lifang Shi, Xia Li, Shaobo Wei, Xiangyuan Ye, Yuan Gao, Yupeng Zhou, Lin Cheng, Long Cheng, Fengying Duan, Mei Li, Hui Zhang, Qian Qian, Wenbin Zhou
Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is required for photosynthetic carbon assimilation, as it catalyses the conversion of inorganic carbon into organic carbon. Despite its importance, RuBisCO is inefficient; it has a low catalytic rate and poor substrate specificity. Improving the catalytic performance of RuBisCO is one of the key routes for enhancing plant photosynthesis. As the basic subunit of RuBisCO, RbcS affects the catalytic properties and plays a key role in stabilizing the structure of holoenzyme. Yet, the understanding of functions of RbcS in crops is still largely unknown. Toward this end, we employed CRISPR-Cas9 technology to randomly edit five rbcS genes in rice (OsrbcS1–5), generating a series of knockout mutants. The mutations of predominant rbcS genes in rice photosynthetic tissues, OsrbcS2–5, conferred inhibited growth, delayed heading and reduced yield in the field conditions, accompanying with lower RuBisCO contents and activities and significantly reduced photosynthetic efficiency. The retarded phenotypes were severer caused by multiple mutations. In addition, we revealed that these mutants had fewer chloroplasts and starch grains and a lower sugar content in the shoot base, resulting in fewer rice tillers. Further structural analysis of the mutated RuBisCO enzyme in one rbcs2,3,5 mutant line uncovered no significant differences from the wild-type protein, indicating that the mutations of rbcS did not compromise the protein assembly or the structure. Our findings generated a mutant pool with genetic diversities, which offers a valuable resource and novel insights into unravelling the mechanisms of RuBisCO in rice. The multiplex genetic engineering approach of this study provides an effective and feasible strategy for RuBisCO modification in crops, further facilitate the photosynthesis improvement and sustainable crop production.
{"title":"Genetic engineering of RuBisCO by multiplex CRISPR editing small subunits in rice","authors":"Yujie Zhou, Lifang Shi, Xia Li, Shaobo Wei, Xiangyuan Ye, Yuan Gao, Yupeng Zhou, Lin Cheng, Long Cheng, Fengying Duan, Mei Li, Hui Zhang, Qian Qian, Wenbin Zhou","doi":"10.1111/pbi.14535","DOIUrl":"https://doi.org/10.1111/pbi.14535","url":null,"abstract":"Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is required for photosynthetic carbon assimilation, as it catalyses the conversion of inorganic carbon into organic carbon. Despite its importance, RuBisCO is inefficient; it has a low catalytic rate and poor substrate specificity. Improving the catalytic performance of RuBisCO is one of the key routes for enhancing plant photosynthesis. As the basic subunit of RuBisCO, RbcS affects the catalytic properties and plays a key role in stabilizing the structure of holoenzyme. Yet, the understanding of functions of RbcS in crops is still largely unknown. Toward this end, we employed CRISPR-Cas9 technology to randomly edit five <i>rbcS</i> genes in rice (<i>OsrbcS1</i>–<i>5</i>), generating a series of knockout mutants. The mutations of predominant <i>rbcS</i> genes in rice photosynthetic tissues, <i>OsrbcS2</i>–<i>5</i>, conferred inhibited growth, delayed heading and reduced yield in the field conditions, accompanying with lower RuBisCO contents and activities and significantly reduced photosynthetic efficiency. The retarded phenotypes were severer caused by multiple mutations. In addition, we revealed that these mutants had fewer chloroplasts and starch grains and a lower sugar content in the shoot base, resulting in fewer rice tillers. Further structural analysis of the mutated RuBisCO enzyme in one <i>rbcs2,3,5</i> mutant line uncovered no significant differences from the wild-type protein, indicating that the mutations of <i>rbcS</i> did not compromise the protein assembly or the structure. Our findings generated a mutant pool with genetic diversities, which offers a valuable resource and novel insights into unravelling the mechanisms of RuBisCO in rice. The multiplex genetic engineering approach of this study provides an effective and feasible strategy for RuBisCO modification in crops, further facilitate the photosynthesis improvement and sustainable crop production.","PeriodicalId":221,"journal":{"name":"Plant Biotechnology Journal","volume":"16 1","pages":""},"PeriodicalIF":13.8,"publicationDate":"2024-12-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142763266","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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