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<front>
<journal-meta>
<journal-id journal-id-type="publisher-id">Front. Plant Sci.</journal-id>
<journal-title>Frontiers in Plant Science</journal-title>
<abbrev-journal-title abbrev-type="pubmed">Front. Plant Sci.</abbrev-journal-title>
<issn pub-type="epub">1664-462X</issn>
<publisher>
<publisher-name>Frontiers Media S.A.</publisher-name>
</publisher>
</journal-meta>
<article-meta>
<article-id pub-id-type="doi">10.3389/fpls.2024.1336726</article-id>
<article-categories>
<subj-group subj-group-type="heading">
<subject>Plant Science</subject>
<subj-group>
<subject>Original Research</subject>
</subj-group>
</subj-group>
</article-categories>
<title-group>
<article-title>Establishment and application of a root wounding&#x2013;immersion method for efficient virus-induced gene silencing in plants</article-title>
</title-group>
<contrib-group>
<contrib contrib-type="author" equal-contrib="yes">
<name>
<surname>Li</surname>
<given-names>Xinyun</given-names>
</name>
<xref ref-type="aff" rid="aff1">
<sup>1</sup>
</xref>
<xref ref-type="author-notes" rid="fn003">
<sup>&#x2020;</sup>
</xref>
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<contrib contrib-type="author" equal-contrib="yes">
<name>
<surname>Tao</surname>
<given-names>Na</given-names>
</name>
<xref ref-type="aff" rid="aff2">
<sup>2</sup>
</xref>
<xref ref-type="author-notes" rid="fn003">
<sup>&#x2020;</sup>
</xref>
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</contrib>
<contrib contrib-type="author">
<name>
<surname>Xu</surname>
<given-names>Bin</given-names>
</name>
<xref ref-type="aff" rid="aff1">
<sup>1</sup>
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<contrib contrib-type="author">
<name>
<surname>Xu</surname>
<given-names>Junqiang</given-names>
</name>
<xref ref-type="aff" rid="aff1">
<sup>1</sup>
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<contrib contrib-type="author">
<name>
<surname>Yang</surname>
<given-names>Zhengan</given-names>
</name>
<xref ref-type="aff" rid="aff1">
<sup>1</sup>
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<contrib contrib-type="author">
<name>
<surname>Jiang</surname>
<given-names>Caiqian</given-names>
</name>
<xref ref-type="aff" rid="aff1">
<sup>1</sup>
</xref>
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<contrib contrib-type="author">
<name>
<surname>Zhou</surname>
<given-names>Ying</given-names>
</name>
<xref ref-type="aff" rid="aff1">
<sup>1</sup>
</xref>
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<contrib contrib-type="author">
<name>
<surname>Deng</surname>
<given-names>Minghua</given-names>
</name>
<xref ref-type="aff" rid="aff1">
<sup>1</sup>
</xref>
<uri xlink:href="https://loop.frontiersin.org/people/344390"/>
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</contrib>
<contrib contrib-type="author" corresp="yes">
<name>
<surname>Lv</surname>
<given-names>Junheng</given-names>
</name>
<xref ref-type="aff" rid="aff1">
<sup>1</sup>
</xref>
<xref ref-type="author-notes" rid="fn001">
<sup>*</sup>
</xref>
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<contrib contrib-type="author" corresp="yes">
<name>
<surname>Zhao</surname>
<given-names>Kai</given-names>
</name>
<xref ref-type="aff" rid="aff1">
<sup>1</sup>
</xref>
<xref ref-type="author-notes" rid="fn001">
<sup>*</sup>
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<aff id="aff1">
<sup>1</sup>
<institution>Key Laboratory of Vegetable Biology of Yunnan Province, College of Landscape and Horticulture, Yunnan Agricultural University</institution>, <addr-line>Kunming, Yunnan</addr-line>, <country>China</country>
</aff>
<aff id="aff2">
<sup>2</sup>
<institution>College of Agronomy and Biotechnology, Yunnan Agricultural University</institution>, <addr-line>Kunming</addr-line>, <country>China</country>
</aff>
<author-notes>
<fn fn-type="edited-by">
<p>Edited by: Xingguo Ye, Chinese Academy of Agricultural Sciences, China</p>
</fn>
<fn fn-type="edited-by">
<p>Reviewed by: Yang Jian, Ningbo University, China</p>
<p>Jinping Zhao, Texas A and M University, United States</p>
</fn>
<fn fn-type="corresp" id="fn001">
<p>*Correspondence: Junheng Lv, <email xlink:href="mailto:junhenglv@ynau.edu.cn">junhenglv@ynau.edu.cn</email>; Kai Zhao, <email xlink:href="mailto:kailixian1023@aliyun.com">kailixian1023@aliyun.com</email>
</p>
</fn>
<fn fn-type="equal" id="fn003">
<p>&#x2020;These authors have contributed equally to this work</p>
</fn>
</author-notes>
<pub-date pub-type="epub">
<day>19</day>
<month>04</month>
<year>2024</year>
</pub-date>
<pub-date pub-type="collection">
<year>2024</year>
</pub-date>
<volume>15</volume>
<elocation-id>1336726</elocation-id>
<history>
<date date-type="received">
<day>11</day>
<month>11</month>
<year>2023</year>
</date>
<date date-type="accepted">
<day>15</day>
<month>03</month>
<year>2024</year>
</date>
</history>
<permissions>
<copyright-statement>Copyright &#xa9; 2024 Li, Tao, Xu, Xu, Yang, Jiang, Zhou, Deng, Lv and Zhao</copyright-statement>
<copyright-year>2024</copyright-year>
<copyright-holder>Li, Tao, Xu, Xu, Yang, Jiang, Zhou, Deng, Lv and Zhao</copyright-holder>
<license xlink:href="http://creativecommons.org/licenses/by/4.0/">
<p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</p>
</license>
</permissions>
<abstract>
<p>In the post-genomic era, virus-induced gene silencing (VIGS) has played an important role in research on reverse genetics in plants. Commonly used <italic>Agrobacterium</italic>-mediated VIGS inoculation methods include stem scratching, leaf infiltration, use of agrodrench, and air-brush spraying. In this study, we developed a root wounding&#x2013;immersion method in which 1/3 of the plant root (length) was cut and immersed in a tobacco rattle virus (TRV)1:TRV2 mixed solution for 30 min. We optimized the procedure in <italic>Nicotiana benthamiana</italic> and successfully silenced <italic>N. benthamiana</italic>, tomato (<italic>Solanum lycopersicum</italic>), pepper (<italic>Capsicum annuum</italic> L.), eggplant (<italic>Solanum melongena</italic>), and <italic>Arabidopsis thaliana</italic> phytoene desaturase (<italic>PDS</italic>), and we observed the movement of green fluorescent protein (GFP) from the roots to the stem and leaves. The silencing rate of <italic>PDS</italic> in <italic>N. benthamiana</italic> and tomato was 95&#x2013;100%. In addition, we successfully silenced two disease-resistance genes, <italic>SITL5</italic> and <italic>SITL6</italic>, to decrease disease resistance in tomatoes (CLN2037E). The root wounding&#x2013;immersion method can be used to inoculate large batches of plants in a short time and with high efficiency, and fresh bacterial infusions can be reused several times. The most important aspect of the root wounding&#x2013;immersion method is its application to plant species susceptible to root inoculation, as well as its ability to inoculate seedlings from early growth stages. This method offers a means to conduct large-scale functional genome screening in plants.</p>
</abstract>
<kwd-group>
<kwd>root wounding&#x2013;immersion method</kwd>
<kwd>virus-induced gene silencing</kwd>
<kwd>agroinoculation</kwd>
<kwd>tobacco rattle virus</kwd>
<kwd>gene function</kwd>
</kwd-group>
<counts>
<fig-count count="6"/>
<table-count count="1"/>
<equation-count count="0"/>
<ref-count count="63"/>
<page-count count="11"/>
<word-count count="4926"/>
</counts>
<custom-meta-wrap>
<custom-meta>
<meta-name>section-in-acceptance</meta-name>
<meta-value>Plant Biotechnology</meta-value>
</custom-meta>
</custom-meta-wrap>
</article-meta>
</front>
<body>
<sec id="s1" sec-type="intro">
<label>1</label>
<title>Introduction</title>
<p>Virus-induced gene silencing (VIGS) is a reverse genetics tool for effectively silencing target gene expression by using plant antiviral defense mechanisms that inhibit invading viruses (<xref ref-type="bibr" rid="B17">Dommes et&#xa0;al., 2019</xref>). Currently, most functional gene research methods, including chemical mutagenesis, T-DNA insertion, and CRISPR/Cas, are dependent on stably transformed lines and generally require labor-intensive processes such as phenotyping and molecular screening, and several of the methods are inefficient. In some cases, the loss of function of some essential genes causes plant death during the early stages of plant growth, which may limit the application of these methods (<xref ref-type="bibr" rid="B4">Baulcombe, 2004</xref>; <xref ref-type="bibr" rid="B5">Benedito et&#xa0;al., 2004</xref>). In comparison, VIGS is a low-cost and rapid tool, and a large number of plant viruses have been successfully developed as VIGS vectors, including tobacco rattle virus (TRV), tobacco mosaic virus (TMV), potato virus X (PVX), tomato golden mosaic virus (TGMV), and cabbage leaf curl virus (CbLCV) (<xref ref-type="bibr" rid="B11">Burch-Smith et&#xa0;al., 2004</xref>).</p>
<p>Modified VIGS systems have been used in Brassicaceae (<xref ref-type="bibr" rid="B20">Fridborg et&#xa0;al., 2013</xref>; <xref ref-type="bibr" rid="B55">Wang et&#xa0;al., 2013</xref>; <xref ref-type="bibr" rid="B59">Yu et&#xa0;al., 2019</xref>), Solanaceae (<xref ref-type="bibr" rid="B21">Fu et&#xa0;al., 2005</xref>), Gramineae (<xref ref-type="bibr" rid="B6">Bennypaul et&#xa0;al., 2012</xref>; <xref ref-type="bibr" rid="B31">Kang et&#xa0;al., 2013</xref>; <xref ref-type="bibr" rid="B38">Martin et&#xa0;al., 2013</xref>), Cucurbitaceae (<xref ref-type="bibr" rid="B9">Bu et&#xa0;al., 2019</xref>; <xref ref-type="bibr" rid="B33">Liao et&#xa0;al., 2019</xref>), Asteraceae (<xref ref-type="bibr" rid="B15">Deng et&#xa0;al., 2012</xref>), Leguminosae (<xref ref-type="bibr" rid="B23">Ganiger et&#xa0;al., 2013</xref>; <xref ref-type="bibr" rid="B3">Atwood et&#xa0;al., 2014</xref>), Orchidaceae (<xref ref-type="bibr" rid="B27">Hsieh et&#xa0;al., 2013a</xref>, <xref ref-type="bibr" rid="B28">Hsieh et&#xa0;al., 2013b</xref>), and Malvaceae (<xref ref-type="bibr" rid="B24">Gao et&#xa0;al., 2011</xref>; <xref ref-type="bibr" rid="B41">Pang et&#xa0;al., 2013</xref>). The silencing efficiency of VIGS is related to the inoculation method, bacterial suspension concentration, vector selection, and environmental conditions. Virus vector-induced silencing efficiency is associated with host selection. Currently, <italic>Agrobacterium</italic>-mediated inoculation is mainly used for VIGS, and the primary methods for this type of inoculation are injection (<xref ref-type="bibr" rid="B29">Huang et&#xa0;al., 2014</xref>), agrodrench (<xref ref-type="bibr" rid="B44">Ryu et&#xa0;al., 2004</xref>), high-pressure spray (<xref ref-type="bibr" rid="B35">Liu et&#xa0;al., 2002a</xref>), and vacuum infiltration (<xref ref-type="bibr" rid="B57">Yan et&#xa0;al., 2012</xref>). TRV is widely used in VIGS vector construction owing to its high silencing efficiency, long silencing duration, and mild infection symptoms (<xref ref-type="bibr" rid="B16">Dinesh-Kumar et&#xa0;al., 2003</xref>). Studies have confirmed that low temperature and low humidity can increase VIGS silencing efficiency (<xref ref-type="bibr" rid="B22">Fu et&#xa0;al., 2006</xref>; <xref ref-type="bibr" rid="B49">Singh et&#xa0;al., 2018</xref>; <xref ref-type="bibr" rid="B58">Yang et&#xa0;al., 2018</xref>), and TRV-VIGS inoculated through agrodrench application or leaf infiltration can persist for 2 years or more (<xref ref-type="bibr" rid="B47">Senthil-Kumar and Mysore, 2011a</xref>). In agricultural inoculation, the concentration of the infiltration solution also considerably affects gene silencing in VIGS experiments; for example, an OD600 &gt; 1 causes <italic>Nicotiana benthamiana</italic> leaf necrosis. In comparison, the OD600 = 1.5 of <italic>Agrobacterium</italic> results in good infection effects in tomatoes (<xref ref-type="bibr" rid="B46">Senthil-Kumar et&#xa0;al., 2007</xref>).</p>
<p>Several Agrobacterium-mediated inoculation methods have been developed to introduce VIGS and other related applications, such as virus-mediated genome editing (<xref ref-type="bibr" rid="B1">Ali et&#xa0;al., 2015</xref>), into host plants. Among these methods, root inoculation is a promising approach that can be applied to research on root systems and on plant hosts that are resistant to above-ground infection, as well as in other similar cases. TRV vectors can infect plant roots and efficiently express green fluorescent protein (GFP) (<xref ref-type="bibr" rid="B37">MacFarlane and Popovich, 2000</xref>). In addition, one study found that a modified TRV vector showed stronger infectivity and invasiveness in meristems in the rhizosphere than elsewhere (<xref ref-type="bibr" rid="B54">Valentine et&#xa0;al., 2004</xref>). The present study employed a root wounding-immersion method to trigger VIGS with TRV vectors. This method is suitable for <italic>N. benthamiana</italic>, tomato (<italic>Solanum lycopersicum</italic>), eggplant (<italic>Solanum melongena</italic>), pepper (<italic>Capsicum annuum</italic> L.), and <italic>Arabidopsis thaliana</italic>. This study provides a new VIGS inoculation approach for functional research on plant genes.</p>
</sec>
<sec id="s2" sec-type="materials|methods">
<label>2</label>
<title>Materials and methods</title>
<sec id="s2_1">
<label>2.1</label>
<title>Plant materials and growth conditions</title>
<p>Tomato (Micro Tom and CLN2037E), <italic>Arabidopsis thaliana</italic>, <italic>Nicotiana benthamiana</italic>, eggplant (S-34), and pepper (<italic>Capsicum annuum</italic>) were all stored by our group. The seeds were placed in cell culture dishes with moist tissue paper and covered, after which they were incubated at 25&#xb0;C under high humidity for 3 days to stimulate germination. After breaking through the testa, the plants were cultured indoors with 16 h (28&#xb0;C) of light and 8 h of darkness (20&#xb0;C).</p>
</sec>
<sec id="s2_2">
<label>2.2</label>
<title>Plasmid construction</title>
<p>pTRV2-<italic>GFP</italic> was used as a backbone (<xref ref-type="fig" rid="f1">
<bold>Figure&#xa0;1A</bold>
</xref>) and <italic>GFP</italic> and phytoene desaturase (<italic>PDS</italic>) were used as reporter genes. <xref ref-type="bibr" rid="B44">Ryu et&#xa0;al. (2004)</xref> found that TRV2-NbPDS could silence endogenous PDS homologs in tomato, tobacco, and Petunia, showing that the <italic>PDS</italic> gene sequences of these species were conserved. However, TRV2-NbPDS did not inhibit the PDS homologs in pepper, potato, or eggplant (<xref ref-type="bibr" rid="B44">Ryu et&#xa0;al., 2004</xref>). To ensure accurate targeting in different hosts, we designed specific primers for different species to clone 300 bp of <italic>PDS</italic>, <italic>SITLP5</italic>, and <italic>SITLP6</italic> silencing fragments, which were ligated to the binary vector TRV2-<italic>GFP</italic> to generate TRV2-<italic>GFP</italic>-<italic>NbPDS</italic>, TRV2-<italic>GFP</italic>-<italic>SlPDS</italic>, TRV2-<italic>GFP</italic>-<italic>CaPDS</italic>, TRV2-<italic>GFP</italic>-<italic>SmPDS</italic>, TRV2-<italic>GFP</italic>-A<italic>tPDS</italic>, TRV2-<italic>GFP</italic>-<italic>SITLP5</italic>, and TRV2-<italic>GFP</italic>-<italic>SITLP6</italic>. The designed primers are shown in <xref ref-type="supplementary-material" rid="ST1">
<bold>Supplementary Table S1</bold>
</xref>. Electroporation was used to transform <italic>Agrobacterium</italic> GV1301 using the binary vectors pTRV2-<italic>GFP</italic>-<italic>PDS</italic> and pTRV1.</p>
<fig id="f1" position="float">
<label>Figure&#xa0;1</label>
<caption>
<p>Schematic diagram of the root wounding&#x2013;immersion method. <bold>(A)</bold> Construction of the pTRV2-<italic>GFP</italic> backbone; <bold>(B, C)</bold> Schematic of the root wounding&#x2013;immersion process.</p>
</caption>
<graphic mimetype="image" mime-subtype="tiff" xlink:href="fpls-15-1336726-g001.tif"/>
</fig>
</sec>
<sec id="s2_3">
<label>2.3</label>
<title>Development of root wounding&#x2013;immersion method and <italic>GFP</italic> imaging</title>
<p>
<italic>Agrobacterium</italic> GV1301 containing pTRV2-<italic>GFP</italic>-<italic>PDS</italic> and pTRV1 were cultured on LB plates containing kanamycin (50 &#x3bc;g/mL) and rifampin (25 &#x3bc;g/mL) at 28&#xb0;C for 2 days. Positive colonies were selected and cultured overnight in LB broth containing the corresponding antibiotics and shaken at 200 rpm. A NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, New York, NY, USA) was used to determine that the OD600 of the inoculum was &gt;1.</p>
<p>One day before VIGS, 50 &#x3bc;L of the above culture was added to 20 mL of LB medium containing 50 &#x3bc;g/mL kanamycin, 25 &#x3bc;g/mL rifampin, 20 &#x3bc;M acetosyringone, and 10 mM MES and cultured overnight at 28&#xb0;C and 200 rpm. The following day, an infiltration solution (10 mM MgCl<sub>2</sub>, 10 mM MES at pH 5.6, 150 &#x3bc;M acetosyringone) was used to resuspend the culture until OD = 0.8. TRV1 and TRV2-carrying <italic>Agrobacterium</italic> suspensions were cultured in the dark at 28&#xb0;C for 3 h (<xref ref-type="bibr" rid="B61">Zhang and Thomma, 2014</xref>; <xref ref-type="bibr" rid="B39">Meziadi et&#xa0;al., 2016</xref>). For the root wounding&#x2013;immersion procedure, seedlings were first cultured in a tray. To increase the resistance of the plants, seedlings with 3&#x2013;4 real leaves that were 3 weeks old were removed from the soil (including the roots). After pure water was used to remove soil and other impurities from the roots, a disinfected leaf knife was used to remove 1/3 of the root lengthwise. We designed two different immersion protocols: 1) &#x201c; concurrent inoculation,&#x201d; where TRV1 and TRV2 were mixed and the plant was immersed for 30 min; and 2) &#x201c;successive inoculation,&#x201d; where the plant was immersed in TRV1 infiltration solution for 15 min and then transferred to a TRV2 infiltration solution for 15 min of immersion. To determine the effect of temperature on infection efficiency, the cut seedlings were placed in infiltration solutions with different temperature gradients in the two immersion protocols so that the roots were completely immersed. Shaking was carried out every 5 min. After 30 min, the seedlings were transferred to a 50-pore plastic tray containing sterilized soil (peat and vermiculite). After 48 h of dark treatment, the plants were cultured indoors with a 16-h (temperature was the same as immersion) light and 8-h dark (18&#xb0;C) cycle with 35% humidity. Furthermore, we designed experiments with different concentrations and multiple uses of the inoculum. A fresh inoculum of 10 mL was prepared to inoculate 20 tomato plants simultaneously, using the same infection methods as described. A portable light source (LUYOR-3410RB, Luyor, USA) was used for <italic>GFP</italic> imaging.</p>
</sec>
<sec id="s2_4">
<label>2.4</label>
<title>RNA isolation and RT-qPCR analysis</title>
<p>To measure the silencing efficiency in TRV-infected plant leaves, a Huayueyang rapid universal plant RNA extraction kit (Huayueyang Biotechnology Co., Ltd., Beijing, China) was used to extract the total RNA from the leaves 30 days after infection. The quality and quantity of the extracted RNA were confirmed by 1.5% (w/v) agarose gel electrophoresis and a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, New York, NY, USA). Then, 2 &#xb5;g of total RNA was used to synthesize cDNA based on the manufacturer&#x2019;s instructions for the TransScript one-step gDNA removal and cDNA synthesis SuperMix (TransGen, Beijing, China) kits. Primer Premier 6 (Premier, San Francisco, CA, USA) software was used to design specific primers for the segments outside the silencing regions. The species-specific internal reference genes were used as control and the primers were synthesized by Tsingke Biotechnology (Qingke Biotechnology Co., Ltd., Beijing, China). SYBR Green PCR Master Mix (TransGen) was used for RT-qPCR on a CFX96 (Bio-Rad, Hercules, CA, USA). Relative mRNA transcript abundance was calculated for TRV-GFP- and TRV-GFP-PDS-infected plants using the 2<sup>&#x2212;&#x394;&#x394;Ct</sup> method (<xref ref-type="bibr" rid="B62">Zhao et&#xa0;al., 2013</xref>) with uninfected plants as controls. All of the primers used are shown in <xref ref-type="supplementary-material" rid="ST1">
<bold>Supplementary Table S1</bold>
</xref>.</p>
</sec>
<sec id="s2_5">
<label>2.5</label>
<title>Pathogen inoculation and disease resistance identification</title>
<p>The late blight pathogen (<italic>Phytophthora infestans</italic>) was obtained from the Institute of Vegetables and Flowers at the Chinese Academy of Agricultural Sciences (Kunming, Yunnan Province, China). Then, 5 &#xd7; 10<sup>4</sup> sporangia/mL was used to spray six or seven leaves of VIGS-successful and control tomato seedlings. After inoculation, the seedlings were placed in a plant growth room, and 100% relative humidity (RH) and 20 &#xb1; 1&#xb0;C were maintained. The seedlings were grown in the dark for 24 h. Then, the seedlings were grown under a 14 h/10 h light/dark cycle with a relative humidity of 60&#x2013;80% (<xref ref-type="bibr" rid="B30">Jiang et&#xa0;al., 2018</xref>). Seven days after inoculation, the method described by <xref ref-type="bibr" rid="B56">Wang (2003)</xref> was used to calculate the disease severity rating (DSR), which was used as a reference for the resistance level.</p>
</sec>
<sec id="s2_6">
<label>2.6</label>
<title>Chlorophyll content measurement</title>
<p>The chlorophyll measurement was immediately performed after the extract was prepared based on the method of <xref ref-type="bibr" rid="B25">Hajirezaei et&#xa0;al. (2002)</xref>. Then, 50 days after infection, the wild type was used as the control, and 50 mg of plant leaves were harvested and frozen in liquid nitrogen. Thereafter, 0.5 mL of 80% (v/v) acetone/water was used for extraction before 0.5 mL of 100% acetone was used for extraction to ensure that chlorophyll a and b were completely extracted. The samples were incubated at 80&#xb0;C for 60 min, and spectrophotometry was used to measure the total chlorophyll, chlorophyll a, and chlorophyll b at 652, 647, and 664 nm, respectively.</p>
</sec>
<sec id="s2_7">
<label>2.7</label>
<title>Phytoene analysis</title>
<p>The samples were rinsed with ice-cold PBS (0.05 mol/L Tris-HCI, pH 7.4 phosphate buffered saline), and filter paper was used to remove the PBS. Then, 0.1 g of each sample was placed in 5 mL of homogenization tubes. Nine volumes of homogenization medium were added to the homogenization tube based on weight (g):volume (mL) = 1:9. The pestle was moved up and down 10 times in an ice bath to prepare 10% homogenate. Afterward, phytoene was measured using the plant phytoene ELISA Kit (Xingtai Sinobest Biotech Co., Ltd.) according to the manufacturer&#x2019;s instructions.</p>
</sec>
<sec id="s2_8">
<label>2.8</label>
<title>Statistical method</title>
<p>Each experiment contained at least three biological replicates. IBM&#xae; SPSS&#xae; Statistics 24 statistical software (IBM, Chicago, IL, USA) was used for data processing. One-way ANOVA was used for data analysis, and Duncan&#x2019;s test was used for post-hoc analysis. A  p &#x2264; 0.05 was considered significant, and the results are presented as the means &#xb1; SD.</p>
</sec>
</sec>
<sec id="s3" sec-type="results">
<label>3</label>
<title>Results and discussion</title>
<sec id="s3_1">
<label>3.1</label>
<title>Optimization of the root wounding&#x2013;immersion procedure</title>
<p>Current inoculation methods mainly include <italic>Agrobacterium</italic>-mediated injection infiltration and vacuum infiltration. However, injecting <italic>Agrobacterium</italic> cultures into the seedling leaves of plants with tough tissues, such as soya beans and maize, can be challenging. Additionally, the leaves of these plants need to be fully unfolded to ensure successful injection (<xref ref-type="bibr" rid="B42">Ratcliff et&#xa0;al., 2001</xref>). For roots that are susceptible to inoculation and early-growth seedlings, we developed an inoculation method known as root wounding&#x2013;immersion (<xref ref-type="fig" rid="f1">
<bold>Figure&#xa0;1B</bold>
</xref>). To test the feasibility of the root wounding&#x2013;immersion method, we chose PDS as a reporter gene because the leaves of plants in which PDS is silenced tend to show symptoms of photobleaching (<xref ref-type="bibr" rid="B32">Kumagai et&#xa0;al., 1995</xref>; <xref ref-type="bibr" rid="B35">Liu et&#xa0;al., 2002a</xref>). To visualize TRV viral transport from the roots to the above-ground parts of plants, we cloned tobacco <italic>NbPDS</italic> fragments and inserted them into the vector pTRV2-<italic>GFP</italic>. Uninjured tobacco was infected according to the method described in Section 2.3 and the silencing rate was found to be less than 1%, so we used uninoculated plants as controls. The frequency of VIGS was defined as the number of plants that show silencing phenotype (photobleaching) after inoculation with TRV2-<italic>GFP-NbPDS</italic>. In <italic>N. benthamiana</italic>, the ratio of positive silenced plants was 95.8%, and <italic>GFP</italic> insertion did not modify the gene silencing capacity of the TRV vector (<xref ref-type="bibr" rid="B52">Tian et&#xa0;al., 2014</xref>). Under illumination with the portable excitation light source (LUYOR-3410RB), pTRV2-<italic>GFP</italic>-<italic>NbPDS</italic> was transferred from the roots to the stem and leaves (<xref ref-type="fig" rid="f2">
<bold>Figure&#xa0;2F</bold>
</xref>). We designed two different infection methods for different temperatures (<xref ref-type="supplementary-material" rid="ST1">
<bold>Supplementary Table S1</bold>
</xref>): &#x201c;concurrent inoculation&#x201d; and &#x201c;successive inoculation.&#x201d; Photobleaching was observed in <italic>N. benthamiana</italic> plants as early as day 6 (<xref ref-type="fig" rid="f2">
<bold>Figure&#xa0;2A</bold>
</xref>), which was faster than the 7&#x2013;10 days reported in the study of <xref ref-type="bibr" rid="B44">Ryu et&#xa0;al. (2004)</xref>.</p>
<fig id="f2" position="float">
<label>Figure&#xa0;2</label>
<caption>
<p>The VIGS system established through the root-soaking method resulted in plant bleaching. <bold>(A&#x2013;E)</bold> Bleaching symptoms photographed in <italic>Nicotiana benthamiana</italic>, tomato, pepper, eggplant, and <italic>Arabidopsis thaliana</italic>, respectively, on day 30; <bold>(F&#x2013;J)</bold> Green fluorescence of <italic>N. benthamiana</italic>, tomato, pepper, eggplant, and <italic>A. thaliana</italic> on days 7, 7, 10, 12, and 4, respectively.</p>
</caption>
<graphic mimetype="image" mime-subtype="tiff" xlink:href="fpls-15-1336726-g002.tif"/>
</fig>
<p>The data show that the survival rate and silencing rate of concurrent inoculation were slightly higher than those for successive inoculation. For example, at 22&#xb0;C, the survival and silencing rates of concurrent inoculation were both 100%, whereas the survival and silencing rates of successive inoculation were 96% and 92%, respectively. We believe the separate immersion in the TRV1 and TRV2 bacterial suspensions decreased opportunities for both to contact each other, which may decrease endogenous RNA-dependent RNA polymerase (RDRP) activation and the replication and production of virus dsRNA (<xref ref-type="bibr" rid="B18">Donaire et&#xa0;al., 2008</xref>). In addition, decreased TRV1 and TRV2 bacterial suspension immersion duration also decreased opportunities for entry into the plant through the wounded root.</p>
<p>Temperature is a key factor affecting the development of the gene-silencing phenotype of VIGS plants (<xref ref-type="bibr" rid="B51">Szittya et&#xa0;al., 2003</xref>; <xref ref-type="bibr" rid="B22">Fu et&#xa0;al., 2006</xref>; <xref ref-type="bibr" rid="B53">Tuttle et&#xa0;al., 2008</xref>), as most viruses lose their potency, decreasing virus concentration. However, low temperatures (16&#x2013;21&#xb0;C for tomatoes) can promote virus silencing (<xref ref-type="bibr" rid="B51">Szittya et&#xa0;al., 2003</xref>; <xref ref-type="bibr" rid="B49">Singh et&#xa0;al., 2018</xref>). Our data show that an appropriate temperature is beneficial for maximizing VIGS silencing effects. In concurrent inoculation, photobleaching occurred after 6 days at 22&#xb0;C and after 12 days at 26&#xb0;C, and VIGS effects were delayed. The survival and silencing rates are also affected by temperature. The survival and silencing rates of concurrent inoculation at 18&#xb0;C were 72% and 79%, respectively, and the survival and silencing rates at 26&#xb0;C were 60% and 56%, respectively. Because the effect of concurrent inoculation was better than that of successive inoculation, concurrent inoculation was adopted in the following studies.</p>
<p>A suitable concentration of permeable solution is beneficial to gene silencing efficiency in VIGS experiments. The most suitable infiltration solution concentration for <italic>N. benthamiana</italic> is OD600 = 1.0 because an inoculum with OD600 &gt; 1.0 may cause leaf necrosis in <italic>N. benthamiana</italic> (<xref ref-type="bibr" rid="B13">Caplan and Dinesh-Kumar, 2006</xref>), and <xref ref-type="bibr" rid="B16">Dinesh-Kumar et&#xa0;al. (2003)</xref> found that OD600 = 1.5 shows better results in tomatoes. <xref ref-type="bibr" rid="B12">Burch-Smith et&#xa0;al. (2006a)</xref> found that the efficiency is almost 100% when the <italic>A. thaliana</italic> Col-0 ecological TRV-VIGS experiment concentration is OD = 1.5. It is necessary to determine the most favorable TRV-VIGS inoculation concentration, and we used different concentrations for concurrent inoculation at 22&#xb0;C for <italic>N. benthamiana</italic> inoculation (<xref ref-type="table" rid="T1">
<bold>Table&#xa0;1</bold>
</xref>). We found that the survival rate and <italic>PDS</italic> silencing effectiveness are dependent on the concentration of the <italic>Agrobacterium</italic> suspension. When OD = 0.4&#x2013;1.2, the rate of <italic>PDS</italic> silencing was more than 95%. However, the <italic>PDS</italic> silencing rate decreased when the concentration was below 0.4, and necrosis occurred when the concentration was above 1.2. Therefore, we resuspended the bacteria to OD600 = 0.8 in the following studies to ensure a higher survival rate and silencing efficiency while considering the efficient use of the <italic>Agrobacterium</italic> resuspension.</p>
<table-wrap id="T1" position="float">
<label>Table&#xa0;1</label>
<caption>
<p>Optimization of concentrations for the root wounding&#x2013;immersion method.</p>
</caption>
<table frame="hsides">
<thead>
<tr>
<th valign="top" align="left">Concentration (OD600)</th>
<th valign="top" align="left">Time of onset of <break/>photobleaching (days)</th>
<th valign="top" align="left">Survival rate (%)</th>
<th valign="top" align="left">Silencing rate (%)</th>
</tr>
</thead>
<tbody>
<tr>
<td valign="top" align="left">0.1</td>
<td valign="top" align="left">6</td>
<td valign="middle" align="left">97</td>
<td valign="middle" align="left">85</td>
</tr>
<tr>
<td valign="top" align="left">0.2</td>
<td valign="top" align="left">6</td>
<td valign="middle" align="left">95</td>
<td valign="middle" align="left">91</td>
</tr>
<tr>
<td valign="top" align="left">0.4</td>
<td valign="top" align="left">7</td>
<td valign="middle" align="left">95</td>
<td valign="middle" align="left">98</td>
</tr>
<tr>
<td valign="top" align="left">0.6</td>
<td valign="top" align="left">6</td>
<td valign="middle" align="left">97</td>
<td valign="middle" align="left">98</td>
</tr>
<tr>
<td valign="top" align="left">0.8</td>
<td valign="top" align="left">6</td>
<td valign="middle" align="left">100</td>
<td valign="middle" align="left">100</td>
</tr>
<tr>
<td valign="top" align="left">1.0</td>
<td valign="top" align="left">7</td>
<td valign="middle" align="left">97</td>
<td valign="middle" align="left">98</td>
</tr>
<tr>
<td valign="top" align="left">1.2</td>
<td valign="top" align="left">6</td>
<td valign="middle" align="left">91</td>
<td valign="middle" align="left">96</td>
</tr>
<tr>
<td valign="top" align="left">1.4</td>
<td valign="top" align="left">6</td>
<td valign="middle" align="left">86</td>
<td valign="middle" align="left">89</td>
</tr>
</tbody>
</table>
</table-wrap>
<p>Even though root inoculation was performed in the root wounding&#x2013;immersion, the movement of virus to aboveground organs was not delayed in <italic>N. benthamiana</italic>. In contrast, we found that the root wounding&#x2013;immersion method does not require leaf infiltration for every <italic>N. benthamiana</italic> plant, and batch processing can be carried out. This method is highly efficient. In addition, we used our previous bacterial suspension (guaranteed fresh) many times. Results showed that the silencing rate was more than 90% (<xref ref-type="supplementary-material" rid="ST2">
<bold>Supplementary Table S2</bold>
</xref>) even after the TRV1:TRV2 mixed bacterial suspension was used five consecutive times. This avoids the preparation of a large volume of infiltration solution for the treatment of a large quantity of materials and saves time and costs. Our method confirms the hypothesis of <xref ref-type="bibr" rid="B44">Ryu et&#xa0;al. (2004)</xref> that <italic>Agrobacterium</italic>-mediated transformation occurs when there is simulated wounding to roots in growing plants.</p>
</sec>
<sec id="s3_2">
<label>3.2</label>
<title>Application of the root wounding&#x2013;immersion method in <italic>A. thaliana</italic>, tomatoes, eggplants, and pepper</title>
<p>Infection was carried out using concurrent inoculation at a concentration of OD = 0.8 and 22&#xb0;C (<xref ref-type="supplementary-material" rid="ST3">
<bold>Supplementary Table S3</bold>
</xref>). We found that the root wounding&#x2013;immersion method is similarly effective in these five plant species, but the infection efficiency differed between different plant species (<xref ref-type="fig" rid="f2">
<bold>Figures&#xa0;2B&#x2013;E</bold>
</xref>). TRV typically causes mild symptoms within 10 days of inoculation in <italic>N. benthamiana</italic> and tomatoes (<xref ref-type="bibr" rid="B35">Liu et&#xa0;al., 2002a</xref>). The virus spreads from the inoculation site to the developing regions of the plant and triggers PTGS. At 2&#x2013;3 weeks, the virus spreads to the completely open upper leaves (<xref ref-type="bibr" rid="B11">Burch-Smith et&#xa0;al., 2004</xref>). Using the root wounding&#x2013;immersion method, we found that photobleaching occurred 1 week after inoculation. The silencing rate of the tomatoes was 95% (<xref ref-type="supplementary-material" rid="ST3">
<bold>Supplementary Table S3</bold>
</xref>). TRV-mediated VIGS silencing has similarly been used in peppers. Generally, the pale yellow (<italic>rbcS</italic>) and photobleached leaf (<italic>PDS</italic>)-silenced phenotype will become significant 2 weeks after TRV vector transformation (<xref ref-type="bibr" rid="B14">Chung et&#xa0;al., 2004</xref>). We employed the root wounding&#x2013;immersion method, and these phenotypes occurred 24 days after inoculation in peppers. The silencing rate of the tomatoes was 58% (<xref ref-type="supplementary-material" rid="ST3">
<bold>Supplementary Table S3</bold>
</xref>). <xref ref-type="bibr" rid="B34">Liu et&#xa0;al. (2012)</xref> used high-pressure spraying and injection on eggplant plants, and uniform bleaching occurred in the newly formed leaves after 30 days (<xref ref-type="bibr" rid="B34">Liu et&#xa0;al., 2012</xref>). We employed root wounding&#x2013;immersion, and bleaching occurred in eggplants after 29 days and spread from leaf veins to the surrounding tissue (<xref ref-type="fig" rid="f2">
<bold>Figure&#xa0;2D</bold>
</xref>). The silencing rate was 23% (<xref ref-type="supplementary-material" rid="ST3">
<bold>Supplementary Table S3</bold>
</xref>). <italic>A. thaliana</italic> is a model plant in the Brassicaceae family, and VIGS has been used for many gene functional studies (<xref ref-type="bibr" rid="B48">Senthil-Kumar and Mysore, 2011b</xref>). Bleaching occurred on day 5 after inoculation, and the silencing rate was 75%. Even though the root wounding&#x2013;immersion method is suitable for <italic>A. thaliana</italic>, the survival rate was 48%, which is lower than that in the tomatoes, peppers, and eggplants. This may be because <italic>A. thaliana</italic> itself is weak, small, and non-viable when it is transplanted after root wounding&#x2013;immersion treatment.</p>
<p>Under portable light excitation via LUYOR-3410RB illumination, pTRV2-<italic>GFP</italic> moved from the roots to the stems and leaves of the tomatoes, peppers, eggplants, and <italic>A. thaliana</italic> (<xref ref-type="fig" rid="f2">
<bold>Figures&#xa0;2G&#x2013;J</bold>
</xref>). Moreover, we used root wounding&#x2013;immersion on cabbage and cucumbers, but photobleaching was not observed (<xref ref-type="supplementary-material" rid="SF1"><bold>Appendix 1</bold></xref>).</p>
</sec>
<sec id="s3_3">
<label>3.3</label>
<title>Successful application of root wounding&#x2013;immersion in multiple plants</title>
<p>We used RT-qPCR to confirm <italic>PDS</italic> silencing at the molecular level, and showed that the average relative expression level of <italic>PDS</italic> was significantly lower in photobleached <italic>N. benthamiana</italic>, tomato, pepper, eggplant, and <italic>A. thaliana</italic> than in the control group (<xref ref-type="fig" rid="f3">
<bold>Figures&#xa0;3A&#x2013;E</bold>
</xref>). The leaves became white owing to carotenoid deficiency and the photooxidative destruction of chlorophyll after <italic>PDS</italic> silencing (<xref ref-type="bibr" rid="B32">Kumagai et&#xa0;al., 1995</xref>). We measured chlorophyll a, chlorophyll b, and total chlorophyll contents upon the silencing of <italic>N. benthamiana</italic>, tomato, pepper, eggplant, and <italic>A. thaliana</italic>. The chlorophyll a, chlorophyll b, and total chlorophyll contents of <italic>N. benthamiana</italic>, tomato, eggplant, pepper, and <italic>A. thaliana</italic> TRV2-<italic>GFP</italic>-<italic>PDS</italic>-silenced plants decreased by more than 40% compared with those of the control group; specifically, the chlorophyll a, chlorophyll b, and total chlorophyll contents in <italic>N. benthamiana</italic> leaves decreased by more than 90% (<xref ref-type="fig" rid="f4">
<bold>Figures&#xa0;4A&#x2013;E</bold>
</xref>).</p>
<fig id="f3" position="float">
<label>Figure&#xa0;3</label>
<caption>
<p>Virus-induced gene silencing in different plants through the root-soaking method. <bold>(A&#x2013;E)</bold> Real-time fluorescence quantitative PCR measurement of <italic>PDS</italic> expression pattern in <italic>Nicotiana benthamiana</italic>, tomato, pepper, eggplant, and <italic>A. thaliana</italic>. The control expression level was assigned a value of 1. The error bars are the standard deviations of biological triplicates. The asterisk (*) means <italic>P</italic> &lt; 0.05, based on Student&#x2019;s <italic>t</italic>-test.</p>
</caption>
<graphic mimetype="image" mime-subtype="tiff" xlink:href="fpls-15-1336726-g003.tif"/>
</fig>
<fig id="f4" position="float">
<label>Figure&#xa0;4</label>
<caption>
<p>The VIGS system established through the root-soaking method resulted in a decrease in the chlorophyll content of plant leaves. <bold>(A&#x2013;E)</bold> The decreases in chlorophyll a, chlorophyll b, and total chlorophyll contents in <italic>Nicotiana benthamiana</italic>, tomato, pepper, eggplant, and <italic>A. thaliana</italic> TRV2-<italic>PDS</italic>-infected plant leaves were measured. The error bars are the standard deviations of biological triplicates. The asterisk (*) means <italic>P</italic> &lt; 0.05, based on Student&#x2019;s <italic>t</italic>-test.</p>
</caption>
<graphic mimetype="image" mime-subtype="tiff" xlink:href="fpls-15-1336726-g004.tif"/>
</fig>
<p>In transfected plants, <italic>PDS</italic> silencing inhibited the synthesis of carotenoids downstream of phytoene and caused high levels of phytoene (<xref ref-type="bibr" rid="B32">Kumagai et&#xa0;al., 1995</xref>; <xref ref-type="bibr" rid="B22">Fu et&#xa0;al., 2006</xref>). We measured the phytoene levels of five plants and found that phytoene content increased in the silenced plants (<xref ref-type="fig" rid="f5">
<bold>Figures&#xa0;5A&#x2013;E</bold>
</xref>). These results confirm that root wounding&#x2013;immersion can cause the <italic>PDS</italic> gene to be successfully silenced.</p>
<fig id="f5" position="float">
<label>Figure&#xa0;5</label>
<caption>
<p>The VIGS system established through the root-soaking method resulted in an increase in the phytoene content of plant leaves. <bold>(A&#x2013;E)</bold> The phytoene accumulation levels in <italic>Nicotiana benthamiana</italic>, tomato, pepper, eggplant, and <italic>A. thaliana</italic> TRV2-<italic>PDS</italic> infected plant leaves were measured. The error bars are the standard deviations of biological triplicates. The asterisk (*) means <italic>P</italic> &lt; 0.05, based on Student&#x2019;s <italic>t</italic>-test.</p>
</caption>
<graphic mimetype="image" mime-subtype="tiff" xlink:href="fpls-15-1336726-g005.tif"/>
</fig>
</sec>
<sec id="s3_4">
<label>3.4</label>
<title>Application of root wounding&#x2013;immersion in disease resistance</title>
<p>Inoculation of a leaf with a VIGS vector containing a gene fragment leads to the transmission of the virus throughout the entire plant (<xref ref-type="bibr" rid="B10">Burch-Smith et&#xa0;al., 2006b</xref>). To test the reliability of the root wounding&#x2013;immersion method, we silenced two late blight (<italic>P. Infestans</italic>) resistance genes (<italic>SITLP5</italic> and <italic>SITLP6)</italic> in tomatoes in addition to the commonly reported genes chalcone synthase (<italic>CHS</italic>) (<xref ref-type="bibr" rid="B50">Spitzer et&#xa0;al., 2007</xref>), H subunit of magnesium protoporphyrin chelatase (<italic>ChlH</italic>) (<xref ref-type="bibr" rid="B26">Hiriart et&#xa0;al., 2002</xref>), and anthocyanidin synthase (<italic>ANS</italic>) (<xref ref-type="bibr" rid="B45">Senthil-Kumar et&#xa0;al., 2008</xref>). <italic>SITLP5</italic> and <italic>SITLP6</italic> overexpression and knockout increased and decreased late blight resistance, respectively, in tomatoes (<xref ref-type="bibr" rid="B63">Zhu et&#xa0;al., 2021</xref>). The CLN2037 tomato inbred line was generated by selective breeding from <italic>Solanum pimpinellifolium</italic> and contains late blight resistance genes. Thus, it is widely used in disease-resistance selective breeding and mining of disease resistance genes (<xref ref-type="bibr" rid="B40">Nowicki et&#xa0;al., 2012</xref>; <xref ref-type="bibr" rid="B60">Zhang et&#xa0;al., 2013</xref>). We used the root wounding&#x2013;immersion method to inoculate TRV1:TRV2-<italic>GFP</italic>-<italic>SITLP5</italic> and TRV1:TRV2-<italic>GFP</italic>- <italic>SITLP6</italic> in tomato (CLN2037E), and disease symptoms were observed 7 days after <italic>P. infestans</italic> inoculation in <italic>SITLP5</italic>- and <italic>SITLP6</italic>-silenced plants using the method of <xref ref-type="bibr" rid="B63">Zhu et&#xa0;al. (2021)</xref>. RT-qPCR was used to measure <italic>SITLP5</italic> transcript abundance in silenced plants, and the average relative expression level of <italic>SITLP5</italic> decreased by 43% and 84% in the stems and leaves, respectively, in CLN2037E. Moreover, <italic>SITLP6</italic> decreased by 80% and 79% in the stems and leaves, respectively (<xref ref-type="fig" rid="f6">
<bold>Figures&#xa0;6A, B</bold>
</xref>). The DSR of the control plant was 1.6, which was classified as high resistance. After <italic>SITLP5</italic> and <italic>SITLP6</italic> silencing, the plants showed decreased <italic>P. infestans</italic> resistance (<xref ref-type="fig" rid="f6">
<bold>Figures&#xa0;6C, D</bold>
</xref>), and the calculated DSRs were 3.0 and 2.6, respectively, which was considered moderate resistance. These results showed that the root wounding&#x2013;immersion method is similarly suitable for TRV-VIGS in other biological studies.</p>
<fig id="f6" position="float">
<label>Figure&#xa0;6</label>
<caption>
<p>The VIGS system established using the root-soaking method reduced the disease resistance of tomato plants. <bold>(A, B)</bold> Real-time fluorescence quantitative PCR measurement of <italic>SITLP5</italic> and <italic>SITLP6</italic> expression patterns in the stems and leaves of <italic>SITLP5</italic>- and <italic>SITLP6</italic>-silenced plants. The control expression level was assigned a value of 1. <bold>(C, D)</bold> Disease severity and resistance of SITLP5- and SITLP6-silenced plants 7 days after inoculation with pathogens. The error bars are the standard deviations of biological triplicates. The asterisk (*) means <italic>P</italic> &lt; 0.05, based on Student&#x2019;s <italic>t</italic>-test.</p>
</caption>
<graphic mimetype="image" mime-subtype="tiff" xlink:href="fpls-15-1336726-g006.tif"/>
</fig>
</sec>
<sec id="s3_5">
<label>3.5</label>
<title>The root wounding&#x2013;immersion method can be used for large-scale, high-efficiency VIGS experiments in genetic studies</title>
<p>Initially, <xref ref-type="bibr" rid="B43">Ruiz et&#xa0;al. (1998)</xref> used mechanical friction to deliver potato virus X (PVX) RNA obtained from in vitro transcription to plants and successfully obtained an <italic>NbPDS</italic> gene silencing phenotype. However, this method is difficult and cumbersome, and the silencing efficiency is low. Later, stem scratch and agroinfiltration methods were successfully applied to PVX VIGS (<xref ref-type="bibr" rid="B7">Bhaskar et&#xa0;al., 2009</xref>; <xref ref-type="bibr" rid="B19">Du et&#xa0;al., 2014</xref>). Antiviral silencing in some susceptible Solanaceae roots strongly inhibited PVX replication levels (<xref ref-type="bibr" rid="B2">Andika et&#xa0;al., 2015</xref>). <xref ref-type="bibr" rid="B42">Ratcliff et&#xa0;al. (2001)</xref> silenced target genes by injection infiltration and confirmed that TRV has many advantages over PVX, TMV, and TGMV. The TRV vector is able to invade roots and express GFP efficiently, whereas the widely used PVX vector is not (<xref ref-type="bibr" rid="B37">MacFarlane and Popovich, 2000</xref>). <xref ref-type="bibr" rid="B8">Bond and Baulcombe (2015)</xref> used TRV as a VIGS-RdDM silencing vector tool to augment the production of a 24-nt sRNA. The silencing rate of tomatoes when injection is used is 50% (<xref ref-type="bibr" rid="B36">Liu et&#xa0;al., 2002b</xref>). Spraying is also widely used for tomatoes, with a silencing rate of 90% (<xref ref-type="bibr" rid="B35">Liu et&#xa0;al., 2002a</xref>; <xref ref-type="bibr" rid="B16">Dinesh-Kumar et&#xa0;al., 2003</xref>). However, it has many equipment requirements, preventing many laboratories from employing this method. We developed the root wounding&#x2013;immersion method and optimized it. We selected 3-week-old seedlings, removed 1/3 of the root lengthwise, and then immersed it in TRV1 and TRV2 <italic>Agrobacterium</italic> mixtures for 30 min. The <italic>N. benthamiana</italic> silencing rate was 95&#x2013;100%. The root wounding&#x2013;immersion method has similar strengths as using agrodrench. Agrodrench (<xref ref-type="bibr" rid="B44">Ryu et&#xa0;al., 2004</xref>) is a 1:1 mixture of <italic>Agrobacterium</italic> sp. TRV1 and TRV2 that is used to soak the roots, and a 10-mL pipette is then used to aspirate 3&#x2013;5 mL into the crown of the plant. Although this method is simple and fast, it consumes a substantial amount of materials. After the TRV1 and TRV2 mixtures are prepared for the root wounding&#x2013;immersion method, they can be used to immerse hundreds of plants each time. After immersion, a fresh bacterial suspension can be used repeatedly, which can save considerable time and costs. Moreover, 60&#x2013;70% of Solanaceae plants other than <italic>N. benthamiana</italic> have shown the <italic>PDS</italic> silencing phenotype when agrodrench was used. The silencing rate of tomatoes when root wounding&#x2013;immersion was used was 95%. In addition to Solanaceae plants, we found that the root wounding&#x2013;immersion method is similarly suitable for <italic>A. thaliana</italic>.</p>
</sec>
</sec>
<sec id="s4" sec-type="conclusions">
<label>4</label>
<title>Conclusion</title>
<p>In the post-genomic era, the large-scale functional genomics method is essential for converting sequence information to functional information. No recent breakthroughs have been achieved regarding TRV-VIGS. Thus, we developed the root wounding&#x2013;immersion method and optimized the procedure. This is an extremely simple and efficient VIGS method that can be used to process large batches of plants (<xref ref-type="fig" rid="f1">
<bold>Figures&#xa0;1B, C</bold>
</xref>). We selected <italic>PDS</italic> and <italic>GFP</italic> as reporter genes and successfully silenced <italic>N. benthamiana</italic>, tomato, pepper, eggplant, and <italic>A. thaliana</italic>. In particular, the efficiency in <italic>N. benthamiana</italic> and tomato was 95&#x2013;100%. Then, two resistance genes (<italic>SITL5</italic> and <italic>SITL6</italic>) were used to test the reliability of the root wounding&#x2013;immersion method. In contrast to leaf infiltration, spray inoculation, and the use of agrodrench, the root wounding&#x2013;immersion method does not require infiltration of individual leaves or preparation of large amounts of inoculation solution, and it has low laboratory requirements. VIGS experiments can be carried out in large batches and with high efficiency within 30 min. In addition, our method is suitable for plant species that are susceptible to root inoculation and seedling inoculation in early growth stages.</p>
</sec>
<sec id="s5" sec-type="data-availability">
<title>Data availability statement</title>
<p>The original contributions presented in the study are included in the article/<xref ref-type="supplementary-material" rid="SF1">
<bold>Supplementary Material</bold>
</xref>, further inquiries can be directed to the corresponding author/s.</p>
</sec>
<sec id="s6" sec-type="author-contributions">
<title>Author contributions</title>
<p>XL: Writing &#x2013; original draft, Writing &#x2013; review &amp; editing. NT: Writing &#x2013; original draft. BX: Investigation, Writing &#x2013; original draft. JX: Funding acquisition, Writing &#x2013; review &amp; editing. ZY: Funding acquisition, Writing &#x2013; review &amp; editing. CJ: Methodology, Writing &#x2013; review &amp; editing. YZ: Data curation, Writing &#x2013; original draft. MD: Funding acquisition, Project administration, Writing &#x2013; original draft. JL: Conceptualization, Funding acquisition, Investigation, Writing &#x2013; review &amp; editing. KZ: Formal analysis, Investigation, Methodology, Resources, Writing &#x2013; review &amp; editing.</p>
</sec>
</body>
<back>
<sec id="s7" sec-type="funding-information">
<title>Funding</title>
<p>The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This research was funded by the National Natural Science Foundation of China (32160715), the Yunnan Province Basic Research Special Key Project (202301AS070078), the Research and Integrated Applications of Key Technologies in the Standardized Production of Facility Vegetables (202102AE090005), and the Innovation Guidance and Technology-Based Enterprise Cultivation Program (202204BI090006).</p>
</sec>
<sec id="s8" sec-type="COI-statement">
<title>Conflict of interest</title>
<p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p>
</sec>
<sec id="s9" sec-type="disclaimer">
<title>Publisher&#x2019;s note</title>
<p>All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.</p>
</sec>
<sec id="s10" sec-type="supplementary-material">
<title>Supplementary material</title>
<p>The Supplementary Material for this article can be found online at: <ext-link ext-link-type="uri" xlink:href="https://www.frontiersin.org/articles/10.3389/fpls.2024.1336726/full#supplementary-material">https://www.frontiersin.org/articles/10.3389/fpls.2024.1336726/full#supplementary-material</ext-link>
</p>
<supplementary-material xlink:href="Image_1.png" id="SF1" mimetype="image/png"/>
<supplementary-material xlink:href="Table_1.xls" id="ST1" mimetype="application/vnd.ms-excel"/>
<supplementary-material xlink:href="Table_2.xls" id="ST2" mimetype="application/vnd.ms-excel"/>
<supplementary-material xlink:href="Table_3.xls" id="ST3" mimetype="application/vnd.ms-excel"/>
</sec>
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