Dataset Information

Fluorescent Soybean Hairy Root Construction and Its Application in the Soybean-Nematode Interaction: An Investigation.

ABSTRACT: The yield of soybean is limited by the soybean cyst nematode (SCN, Heterodera glycines). Soybean transformation plays a key role in gene function research but the stable genetic transformation of soybean usually takes half a year. Here, we constructed a vector, pNI-GmUbi, in an Agrobacterium rhizogenes-mediated soybean hypocotyl transformation to induce fluorescent hairy roots (FHRs). We describe the operation of FHR-SCN, a fast, efficient and visual operation pathosystem to study the gene functions in the soybean-SCN interaction. With this method, FHRs were detected after 25 days in 4 cultivars (Williams 82, Zhonghuang 13, Huipizhiheidou and Peking) and at least 66.67% of the composite plants could be used to inoculate SCNs. The demographics of the SCN could be started 12 days post-SCN inoculation. Further, GmHS1pro-1 was overexpressed in the FHRs and GmHS1pro-1 provided an additional resistance in Williams 82. In addition, we found that jasmonic acid and JA-Ile increased in the transgenic soybean, implying that the resistance was mainly caused by affecting the content of JA and JA-Ile. In this study, we established a pathosystem, FHR-SCN, to verify the functional genes in soybeans and the SCN interaction. We also verified that GmHS1pro-1 provides additional resistance in both FHRs and transgenic soybeans, and the resistance may be caused by an increase in JA and JA-Ile contents.

SUBMITTER: Yang R

PROVIDER: S-EPMC8699024 | biostudies-literature | 2021 Dec

REPOSITORIES: biostudies-literature

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Fluorescent Soybean Hairy Root Construction and Its Application in the Soybean-Nematode Interaction: An Investigation.

Yang Ruowei R Li Shuang S Yang Xiaowen X Zhu Xiaofeng X Fan Haiyan H Xuan Yuanhu Y Chen Lijie L Liu Xiaoyu X Wang Yuanyuan Y Duan Yuxi Y

Biology 20211220 12

<h4>Background</h4>The yield of soybean is limited by the soybean cyst nematode (SCN, <i>Heterodera glycines</i>). Soybean transformation plays a key role in gene function research but the stable genetic transformation of soybean usually takes half a year.<h4>Methods</h4>Here, we constructed a vector, pNI-GmUbi, in an <i>Agrobacterium rhizogenes</i>-mediated soybean hypocotyl transformation to induce fluorescent hairy roots (FHRs).<h4>Results</h4>We describe the operation of FHR-SCN, a fast, eff ...[more]

PMID: 34943269

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Project description:Root-knot nematodes (RKNs) infect many annual and perennial crops and are the most devastating soil-born pests in vineyards. To develop a biotech-based solution for controlling RKNs in grapes, we evaluated the efficacy of plant-derived RNA interference (RNAi) silencing of a conserved RKN effector gene, 16D10, for nematode resistance in transgenic grape hairy roots. Two hairpin-based silencing constructs, containing a stem sequence of 42 bp (pART27-42) or 271 bp (pART27-271) of the 16D10 gene, were transformed into grape hairy roots and compared for their small interfering RNA (siRNA) production and efficacy on suppression of nematode infection. Transgenic hairy root lines carrying either of the two RNAi constructs showed less susceptibility to nematode infection compared with control. Small RNA libraries from four pART27-42 and two pART27-271 hairy root lines were sequenced using an Illumina sequencing technology. The pART27-42 lines produced hundred times more 16D10-specific siRNAs than the pART27-271 lines. On average the 16D10 siRNA population had higher GC content than the 16D10 stem sequences in the RNAi constructs, supporting previous observation that plant dicer-like enzymes prefer GC-rich sequences as substrates for siRNA production. The stems of the 16D10 RNAi constructs were not equally processed into siRNAs. Several hot spots for siRNA production were found in similar positions of the hairpin stems in pART27-42 and pART27-271. Interestingly, stem sequences at the loop terminus produced more siRNAs than those at the stem base. Furthermore, the relative abundance of guide and passenger single-stranded RNAs from putative siRNA duplexes was largely correlated with their 5' end thermodynamic strength. This study demonstrated the feasibility of using a plant-derived RNAi approach for generation of novel nematode resistance in grapes and revealed several interesting molecular characteristics of transgene siRNAs important for optimizing plant RNAi constructs.

Project description:BackgroundAgrobacterium-mediated genetic transformation is a widely used and efficient technique for gene functional research in crop breeding and plant biology. While in some plant species, including soybean, genetic transformation is still recalcitrant and time-consuming, hampering the high-throughput functional analysis of soybean genes. Thus we pursue to develop a rapid, simple, and highly efficient hairy root system induced by Agrobacterium rhizogenes (A. rhizogenes) to analyze soybean gene function.ResultsIn this report, a rapid, simple, and highly efficient hairy root transformation system for soybean was described. Only sixteen days were required for the whole workflow and the system was suitable for various soybean genotypes, with an average transformation frequency of 58-64%. Higher transformation frequency was observed when wounded cotyledons from 1-day-germination seeds were inoculated and co-cultivated with A. rhizogenes in 1/2 B5 (Gamborg' B-5) medium. The addition of herbicide selection to root production medium increased the transformation frequency to 69%. To test the applicability of the hairy root system for gene functional analysis, we evaluated the protein expression and subcellular localization in transformed hairy roots. Transgenic hairy roots exhibited significantly increased GFP fluorescence and appropriate protein subcellular localization. Protein-protein interactions by BiFC (Bimolecular Fluorescent Complimentary) were also explored using the hairy root system. Fluorescence observations showed that protein interactions could be observed in the root cells. Additionally, hairy root transformation allowed soybean target sgRNA screening for CRISPR/Cas9 gene editing. Therefore, the protocol here enables high-throughput functional characterization of candidate genes in soybean.ConclusionA rapid, simple, and highly efficient A. rhizogenes-mediated hairy root transformation system was established for soybean gene functional analysis, including protein expression, subcellular localization, protein-protein interactions and gene editing system evaluation.

Dataset Information

Fluorescent Soybean Hairy Root Construction and Its Application in the Soybean-Nematode Interaction: An Investigation.

Publications

Fluorescent Soybean Hairy Root Construction and Its Application in the Soybean-Nematode Interaction: An Investigation.

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