这项工作部分得到了瓦格宁根大学基金（WUF）、中国研究生奖学金理事会计划以及NWO-VIDI赠款12378的支持。

1. 植物生长和测试条件
<ol><li>在18-22 °C的温度范围内或在受控温室或气候室中种植和维持植物，并在自然光照照下或以16小时/8小时的昼夜为系统。去除辅助分支，使植物更容易管理。</li>
	<li>对于马铃薯，在无菌塑料罐 中保持体外 植物，其中含有 村希格-斯库格 （MS）介质（20克/升蔗糖）， 5 g/L MS 含维生素的盐，8 克/升，pH 5.8），在 18 °C 的气候室受控条件下，使用 16 小时/8 小时的日夜制度，持续两周，然后将其转移到受管制温室的消毒土壤盆中。</li>
</ol>2. 农业渗透
<ol><li>植物材料： 对于 尼科蒂亚娜本塔米亚纳，使用约4-5周大的种子生长植物。对于马铃薯，使用约4-5周大的 体外移植。选择年轻、健康、发育的叶子进行渗透。</li>
	<li>
农用细菌 文化：<ol>
<li>提前准备 YEB 介质（表 1）。用 10 毫升 YEB 介质填充 50 毫升管子，辅以 1μl 乙毒素 （200 mM）、100μl MES 缓冲器 （2-（N-莫索利诺）-硫酸乙烷、1 M） 和适当的抗生素。皮佩特 20μl 甘油库存所需的菌株 （表2） （包含感兴趣的基因） 进入 YEB.同时为此测定中的所有 农用细菌 菌株启动培养。在 28 °C 和 200 rpm 下摇动 1-2 天，孵化细胞，以约1.0 的 OD 600 进行孵化。</li>
		<li>通过离心在3，000 x g下收获细胞10分钟。倒掉超自然物，用新制作的MMA介质（表3）将颗粒重新注入0.3的OD600。 轻轻涡流细胞以恢复它们。对于两种细菌菌株的硬币渗透，将培养物按1：1的比例混合。</li>
		<li>在渗透前，在室温下将培养物放在长凳上1-6小时。同时，给被渗透的植物贴上标签，并标注实验日期。</li>
	</ol>
</li>
	<li>渗透： 使用1毫升无针注射器渗透 到农用细菌 悬架中。小心而缓慢地将叶板与注射器上的悬架一起注射（出于健康和安全原因，在此过程中应佩戴眼睛保护）。每个菌株至少渗透三株植物。每株使用三片叶子作为三叶。 注：通常，1毫升 的农用细菌 悬浮剂可能足够3株 N.本塔米亚纳。对于马铃薯，需要更多的悬浮，因为 索拉努姆 物种的叶子更难渗透。所需的悬浮量取决于 索拉努姆 物种。通过更换手套或在渗透之间用乙醇消毒手套，以及直到接种后的第二天才为植物浇水，避免交叉污染。</li>
	<li>得分： 当细胞死亡表型清晰时，在渗透后3天左右的评分宏观反应（图1）。如果细胞死亡表型是定量的，请使用给定标准（图 2）。简言之，细胞死亡的宏观评分比例主要取决于渗透区域的细胞死亡百分比。细胞死亡的百分比从0%（无症状）到100%（细胞死亡）。中间反应范围从微弱反应，如氯化症到细胞死亡水平的增加。如有需要，还可以使用现代光像设备对宏观评分进行定量评估。</li>
</ol>3. PVX 农业感染
<ol><li>植物材料： 对于 N.本塔米亚纳，使用大约2-3周大的种子生长的植物。对于马铃薯，使用大约2-3周大的 体外移植。对于大规模测试，使用稍老（4-5周）的植物，因为这些植物有越来越多的叶子，以适应更多的 农用细菌 接种点。</li>
	<li>
农用细菌 培养：<ol>
<li>提前准备 YEB 介质。用3毫升YEB填充10毫升管，并辅以适当的抗生素。皮佩特 20μl 甘油库存所需的菌株 （表2） （包含感兴趣的基因） 进入 YEB.同时为此测定中的所有 农用细菌 菌株启动培养。在 28 °C 和 200 rpm 下摇动 1-2 天，孵化细胞，以约1.0 的 OD 600 进行孵化。</li>
		<li>每个 农用细菌 菌株约300μl，并将其传播到LB固体阿加中板，辅以适当的抗生素和孵育细胞在28°C 1-2天。</li>
	</ol>
</li>
	<li>感染： 用铲子收集盘子里的 农用细菌 培养物。在刮刀上浸入 农用细菌 培养物中的木制牙签，刺穿叶子以接种大量细菌。为每株至少接种三株植物。每株使用三片叶子作为三叶。在每片叶子中，为每株进行多个接种位点。</li>
	<li>得分： 接种后两周左右的宏观反应得分（图3）。对于高通量屏幕，记录每个接种点的定性响应（是/否）。然后计算响应站点的百分比，并将它们与控件进行比较。</li>
</ol>

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作者宣称他们没有相互竞争的经济利益。

农业渗透和农用感染等瞬态检测是现代分子植物病理学研究的重要有效方法。尽管存在一些限制，但这些方法满足了工厂对高效、稳健的高通量功能分析的需求。
农用渗透系统是一种在一系列植物物种中广泛使用的功能检测。农业渗透促进将几个转基因基因同时表达相互作用的蛋白质输送到同一细胞中。这有利于通过硬币渗透农用菌株来概括R-AVR关系，用表达匹配R基因的菌株来表达AVR基因。此外，对于已知的R-AVR对，此类硬币胶片可用作正控件。包括这种控制很重要，因为在某些植物基因型中，转化效率可能低于检测响应的阈值。包括负对照，例如含有没有插入基因的载体的农用细菌菌株，对于确定某种植物基因型是否对农用细菌产生非特异性防御反应也至关重要。此功能在马铃薯种质中以一定频率发生，并非所有索拉努姆物种都非常适合这种基于农用细菌的表达系统。一般来说，农用渗透检测在N.本塔米亚纳和大多数马铃薯基因型中效果很好。除了效应学之外，农业渗透技术还有其他各种潜在应用，例如通过共聚焦显微镜从转基因中产生蛋白质和植物细胞中的蛋白质本地化。
PVX 农业感染是一种高度敏感的筛查系统，通常更适合高通量筛查。由于 农用细菌 现在只存在于本地，因此，由于PVX病毒接管了转基因的进一步传播，对这种细菌的非特异性反应现在不是很令人不安。然而，植物可能对PVX具有抗药性，或具有极端抗药性（ER）反应，在这种情况下，农业感染方法不合适。PVX农用感染方法的另一个局限性是兴趣基因的插入大小。观察到的反应表型可能因伤口周围强烈的黑色坏死而不同，到接种点附近的微弱坏死。在 N.本塔米亚纳 和 索拉努姆 物种中，PVX农业感染被公认为比农业渗透更敏感。
考虑到不同测试植物基因型的遗传背景可能有一些限制（见上文），我们通常通过PVX农业感染和农业渗透得出类似的结论。这些结果与其他检测结果（如蛋白质渗透29 和ELISA3）也相当。考虑到两个系统的优点和局限性，我们建议使用这两种方法来补充或确认独立的结果。

效应学，一种高通量功能基因组学方法最近已成为识别作物中抗药性（R）基因和匹配病原体1-4的avirence（Avr）基因的有力工具。与R基因更耗时的稳定转化相比，效应定向策略基于病原体基因序列的瞬时检测。
自基因组学时代以来，植物病原体的基因组得到了广泛的探索。例如，对于包括最具破坏性的植物病原体在内的卵母细胞，已经生成并分析了大量序列，以寻找在与植物5-10相互作用期间发挥作用的基因。一类病原蛋白代表效应器，它操纵宿主细胞结构和功能，或促进感染（毒性因素）或触发防御反应（病毒因子）11-13。含有R基因的植物细胞中AVR基因的表达通常会导致超敏感细胞死亡反应（HR）14，15。在植物表达的R和Avr基因可以完成使用瞬态表达系统，如农用细菌的图姆法西恩为基础的瞬态转化（农业渗透）16。这种瞬态转化也可以与病毒表达系统（农业感染）17，18相结合应用。
对于农业过滤，最常用的剂是 A.tmefaciens， 一种广泛的多能植物的宿主范围病原体。 A. 肿瘤 含有肿瘤诱导（Ti）质粒。在细菌的毒力机制被激活后，从Ti质粒转移DNA（T-DNA）将转移到植物细胞中。这可以在受伤的植物细胞中触发，由释放的低分子量酚类化合物和单糖在微酸环境中19。在 农用细菌 悬浮物渗透到主要静脉定义的叶板中后，毒性基因被激活。然后，叶板中的植物细胞将被转化，并表达T-DNA区域所含的转基因。
农业感染是基于伤口接种的农用细菌，它调解病毒转移到植物细胞。然后，在没有农用细菌的情况下，病毒进一步扩散到邻近的植物组织。对于农业感染，可以使用多种植物病毒。RNA病毒是基因表达的理想载体，因为它们可以在受感染的植物中繁殖到非常高的水平。在植物RNA病毒中，马铃薯病毒X（PVX）被广泛用于效应学屏幕。为了便于插入基因的功能测试，含有PVX基因组的二元载体被克隆成A.tumefaciens20的T-DNA。。T-DNA被转移到植物细胞后，T-DNA中所含的PVX基因组从35S促进者转录。然后病毒颗粒在受感染的植物中系统地传播，导致插入基因的表达。这种基于农用细菌和PVX的方法称为PVX农业感染。
在这里，我们展示了农业渗透和PVX农业感染检测的例子。作为宿主植物，我们使用马铃薯种质（索拉努姆部分佩托塔），为此，效应学方法已被开创和证明成功3，4。我们还使用尼科蒂亚娜本塔米亚纳，这是著名的模型工厂在索拉纳塞斯植物14，21，22。

<table border="0" cellpadding="0" cellspacing="0" width="955">
	<colgroup>
		<col />
		<col />
		<col span="2" />
	</colgroup>
	<tbody>
		<tr height="27">
			<td height="27" style="height:27px;width:252px;">Beef extract</td>
			<td style="width:267px;">Sigma-Aldrich</td>
			<td style="width:219px;">B4888</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:252px;">Bacteriological peptone</td>
			<td style="width:267px;">Oxoid</td>
			<td style="width:219px;">LP0037</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:252px;">Yeast extract</td>
			<td style="width:267px;">Oxoid</td>
			<td style="width:219px;">LP0021</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:252px;">MgSO4</td>
			<td style="width:267px;">Sigma-Aldrich</td>
			<td style="width:219px;">208094</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:252px;">MS salts (without vitamins)</td>
			<td style="width:267px;">Duchefa Biochemie</td>
			<td style="width:219px;">M0221</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:252px;">MES</td>
			<td style="width:267px;">Duchefa Biochemie</td>
			<td style="width:219px;">M1503</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:252px;">LB broth powder</td>
			<td style="width:267px;">Sigma-Aldrich</td>
			<td style="width:219px;">L3022</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:252px;">Acetosyringone</td>
			<td style="width:267px;">Sigma-Aldrich</td>
			<td style="width:219px;">D134406</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:252px;">Syringe (1 ml)</td>
			<td style="width:267px;">BD Plastipak</td>
			<td style="width:219px;">300013</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:252px;">Incubator</td>
			<td style="width:267px;">Infors HT</td>
			<td style="width:219px;">Multitron II</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:252px;">Centrifuge</td>
			<td style="width:267px;">Heraeus</td>
			<td style="width:219px;">Multifuge 3S-R</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:252px;">Spectrophotometer</td>
			<td style="width:267px;">Eppendorf</td>
			<td style="width:219px;">Biophotometer 6131</td>
			<td style="width:219px;"></td>
		</tr>
	</tbody>
</table>

agroinfiltration and pvx agroinfection in potato and nicotiana benthamiana

农业渗透和PVX农业感染是植物候选基因功能分析的两种有效的瞬态表达检测。农用渗透最常用的剂是 农用细菌， 这是许多迪科特植物物种的病原体。这意味着农业渗透可以应用于许多植物物种。在这里，我们介绍我们的协议和预期的结果时，应用这些方法的马铃薯（索拉努姆管），其相关的野生块茎轴承 索拉努姆 物种（索拉努姆 节 佩托塔）和模型植物 尼科蒂亚娜本塔米亚纳。除了对单个基因（如抗药性（R）或抗病毒（Avr）基因）进行功能分析外，农用渗透检测非常适合通过简单地将 R 和 Avr 转基因引入同一细胞来回顾与特定宿主病原体相互作用相关的 R-AVR 相互作用。然而，一些植物基因型可以提高非特异性防御反应 的农用细菌，正如我们观察到的，例如几个马铃薯基因型。与农业渗透相比，PVX农用感染的AVR活性检测更敏感，功能屏幕的吞吐量更高，对 农用细菌的非特异性防御反应的敏感度较低。但是，PVX 可能发生非特定防御，并且由于病毒引起的极端耐药性而有可能错过响应。尽管有这些限制，但根据我们的经验，农业渗透和PVX农业感染都是适当和互补的检测，可以同时用于确认彼此的结果。

图1显示了马铃薯和N.本塔米亚纳的7种不同效应剂（E1-E7）的农业渗透有代表性的实验。细胞死亡出现在渗透后约3天的渗透叶板。细胞死亡表型的程度需要与控制器进行比较。农用细菌株AGL1（pVirG）23的混合物含有双增生-R3a和pK7WG2-AVR3a用作正对照24，25，而 AGL1（pVirG） 用作负控制。图1A和1B分别显示了马铃薯基因型MaR8（马斯滕布罗克R8）26和N.本塔米亚纳的农业过滤的良好例子，它们非常适合农业过滤。叶板中含有与阳性控制相同的细胞死亡，而负控制或 pBINplus-R3a 则不会发生细胞死亡反应。在MaR8中，两个效应器AVR3a（E2）和AVR4（E3）诱发细胞死亡，而其他效应器（E1和E4-E7）则不会。 在N.本塔米亚纳，没有一个测试的效应器能诱导细胞死亡反应。图1C和1D显示了野生马铃薯索拉努姆泊位483-1和索拉努姆雷切210-5的农业渗透的例子，这些例子对农用渗透技术不太适应。在S.伯斯托蒂483-1中，叶组织对负控件以及所有测试的效应器表现出非特异性坏死。在S.rechei 210-5中，渗透的叶板显示细胞死亡对阳性控制的反应非常微弱。
图2 显示了一系列的评分尺度，可用于量化对农业渗透 性农用细菌的反应。细胞死亡的百分比以0-100%的比例描述。观察到的表型范围从宏观上不可见的症状（0%），到显示氯化和细胞死亡水平增加的一系列中间反应，最高可达汇合细胞死亡（100%）。
图3显示了马铃薯PVX农用感染后的一个具有代表性的实验。通常，在拔牙接种后两周左右，这些部位可以发现细胞死亡扩大。如两个图面板所示，扩大细胞死亡存在于用pGR106-CRN2（使用效应器皱褶器Crn2进行正控制）的拔牙接种部位，这是来自P.侵扰者27的一般细胞死亡诱导基因。除了对伤害的轻微反应外，在用pGR106空（负控制）接种牙齿的部位没有注意到细胞死亡扩大。在图3A中，代表马铃薯基因型MaR3（马斯滕布鲁克R3），两个pGR106效应器（E1和E2）没有诱发细胞死亡。在图3B中，介绍了在野生索拉努姆万坎本斯354-1的效果筛选的阳性结果：细胞死亡反应观察到两个pGR106效应器（E1-E3，代表引出）28。
<img alt="Figure 1" fo:content-width="6in" fo:src="/files/ftp_upload/50971/50971fig1highres.jpg" src="/files/ftp_upload/50971/50971fig1.jpg"> 图1。马铃薯和本塔米亚纳的农业渗透的例子。植物包括 （A） 马铃薯基因型马 R8 （马斯滕布罗克 R8）， （B） N. 本塔米亚纳， （C）索拉努姆伯托尔蒂483-1 和 （D）索拉努姆雷切210-5 被渗透与混合物农用菌株AGL1（pVirG）23含有双增-R3a和pK7WG2-AVR3a（正控制 ）、AGL1（pvirG）（ 负控制）、双增-R3a、 和七个效应器（E1-E7）。<a href="https://www.jove.com/files/ftp_upload/50971/50971fig1highres.jpg" target="_blank">单击此处查看更大的图像</a>。
<img alt="Figure 2" fo:content-width="6in" fo:src="/files/ftp_upload/50971/50971fig2highres.jpg" src="/files/ftp_upload/50971/50971fig2.jpg"> 图2。农业渗透反应量化。照片显示细胞死亡的代表性评分表，从0%（无症状）到100%（共生细胞死亡）不等。中间反应范围从微弱反应，如氯化症到细胞死亡水平的增加。 <a href="https://www.jove.com/files/ftp_upload/50971/50971fig2highres.jpg" target="_blank">单击此处查看更大的图像</a>。
<img alt="Figure 3" fo:content-width="5in" fo:src="/files/ftp_upload/50971/50971fig3highres.jpg" src="/files/ftp_upload/50971/50971fig3.jpg"> 图3。马铃薯中PVX农业感染的例子。马铃薯基因型 MaR3 （马斯滕布罗克 R3）（A）和 索拉努姆万卡班本斯 354-1（B） 拔牙接种 pGR106-CRN2 （正控制）， pGR106 空 （负控制） 和 pGR106-E1-2 （效果）， 或 pGR106-E1-E3， <a href="https://www.jove.com/files/ftp_upload/50971/50971fig3highres.jpg" target="_blank">单击此处查看更大的图像</a>。
表1。叶布介质
<table border="1"><tbody>
<tr>
<td>1 L</td>
			<td>蒸馏H2O</td>
		</tr>
<tr>
<td>5 克</td>
			<td>蔗糖</td>
		</tr>
<tr>
<td>5 克</td>
			<td>牛肉提取物</td>
		</tr>
<tr>
<td>5 克</td>
			<td>细菌肽</td>
		</tr>
<tr>
<td>1 克</td>
			<td>酵母提取物</td>
		</tr>
<tr>
<td>2 毫升</td>
			<td>MgSO4 (1 M)</td>
		</tr>
</tbody></table>表2。用于农业渗透和PVX农业感染的载体和菌株。可以使用多个二进制向量。下面列表中的载体允许高表达候选基因，并且在我们手中运作良好。我们更喜欢使用农用菌株GV3101（pMP90）29在N.本塔米亚纳，并发现AGL130含有帮助质粒pVirg（pBBR1MCS-5.virGN54D）23更适合土豆31。其他组已经分析了各种模型植物的其他菌株，包括N.本塔米亚纳，但不是在马铃薯32。
<img alt="Table 2" fo:content-width="5in" fo:src="/files/ftp_upload/50971/50971table2highres.jpg" src="/files/ftp_upload/50971/50971table2.jpg">
GW： 网关版本36
表3。妈妈介质。 <img alt="Table 3" fo:content-width="5in" fo:src="/files/ftp_upload/50971/50971table3highres.jpg" src="/files/ftp_upload/50971/50971table3.jpg">
将pH口调整为5.6。

Watch this Scientific Journal Video about Agroinfiltration and PVX Agroinfection in Potato and Nicotiana benthamiana at JoVE.com

Agroinfiltration and PVX Agroinfection in Potato and Nicotiana benthamiana

农业渗透和PVX农业感染是植物中基因瞬时异位表达的常规功能检测。这些方法是有效电理学策略（快速抗药性和抗病毒基因发现）的有效分析，对分子植物病理学的现代研究至关重要。它们满足了工厂对强健的高通量功能分析的需求。

马铃薯和尼科蒂亚娜本塔米亚纳的农业渗透和PVX农业感染

Agroinfiltration and PVX agroinfection are two efficient transient expression assays for functional analysis of candidate genes in plants. The most ...

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Agroinfiltration and PVX Agroinfection in Potato and Nicotiana benthamiana

20.1K 次观看.  Wageningen University. 农业渗透和PVX农业感染是植物中基因瞬时异位表达的常规功能检测。这些方法是有效电理学策略（快速抗药性和抗病毒基因发现）的有效分析，对分子植物病理学的现代研究至关重要。它们满足了工厂对强健的高通量功能分析的需求。

视频: Agroinfiltration and PVX Agroinfection in Potato and Nicotiana benthamiana

Virus-induced Gene Silencing (VIGS) in Nicotiana benthamiana and Tomato

RNA interference (RNAi) is a highly specific gene-silencing phenomenon triggered by dsRNA1. This silencing mechanism uses two major classes of RNA regulators: microRNAs, which are produced from non-protein coding genes and short interfering RNAs (siRNAs). Plants use RNAi to control transposons and to exert tight control over developmental processes such as flower organ formation and leaf development2,3,4. Plants also use RNAi to defend themselves against infection by viruses. Consequently, many viruses have evolved suppressors of gene silencing to allow their successful colonization of their host5.
Virus-induced gene silencing (VIGS) is a method that takes advantage of the plant RNAi-mediated antiviral defense mechanism. In plants infected with unmodified viruses the mechanism is specifically targeted against the viral genome. However, with virus vectors carrying sequences derived from host genes, the process can be additionally targeted against the corresponding host mRNAs. VIGS has been adapted for high-throughput functional genomics in plants by using the plant pathogen Agrobacterium tumefaciens to deliver, via its Ti plasmid, a recombinant virus carrying the entire or part of the gene sequence targeted for silencing. Systemic virus spread and the endogenous plant RNAi machinery take care of the rest. dsRNAs corresponding to the target gene are produced and then cleaved by the ribonuclease Dicer into siRNAs of 21 to 24 nucleotides in length. These siRNAs ultimately guide the RNA-induced silencing complex (RISC) to degrade the target transcript2.
Different vectors have been employed in VIGS and one of the most frequently used is based on tobacco rattle virus (TRV). TRV is a bipartite virus and, as such, two different A. tumefaciens strains are used for VIGS. One carries pTRV1, which encodes the replication and movement viral functions while the other, pTRV2, harbors the coat protein and the sequence used for VIGS6,7. Inoculation of Nicotiana benthamiana and tomato seedlings with a mixture of both strains results in gene silencing. Silencing of the endogenous phytoene desaturase (PDS) gene, which causes photobleaching, is used as a control for VIGS efficiency. It should be noted, however, that silencing in tomato is usually less efficient than in N. benthamiana. RNA transcript abundance of the gene of interest should always be measured to ensure that the target gene has efficiently been down-regulated. Nevertheless, heterologous gene sequences from N. benthamiana can be used to silence their respective orthologs in tomato and vice versa8.

Virus-induced Gene Silencing VIGS in Nicotiana benthamiana and Tomato

Description of a virus-induced gene silencing (VIGS) method for knock-down of gene expression in Nicotiana benthamiana and tomato.

病毒诱导的基因沉默（VIGS）烟草benthamiana和番茄

RNA interference (RNAi) is a highly specific gene-silencing phenomenon triggered by dsRNA1. This silencing mechanism uses two major classes of RNA ...

科研

生物学

Efficient Agroinfiltration of Plants for High-level Transient Expression of Recombinant Proteins

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing worldwide demand for pharmaceuticals due to its limited scalability and high cost. Plants have shown to be one of the most promising alternative pharmaceutical production platforms that are robust, scalable, low-cost and safe. The recent development of virus-based vectors has allowed rapid and high-level transient expression of recombinant proteins in plants. To further optimize the utility of the transient expression system, we demonstrate a simple, efficient and scalable methodology to introduce target-gene containing Agrobacterium into plant tissue in this study. Our results indicate that agroinfiltration with both syringe and vacuum methods have resulted in the efficient introduction of Agrobacterium into leaves and robust production of two fluorescent proteins; GFP and DsRed. Furthermore, we demonstrate the unique advantages offered by both methods. Syringe infiltration is simple and does not need expensive equipment. It also allows the flexibility to either infiltrate the entire leave with one target gene, or to introduce genes of multiple targets on one leaf. Thus, it can be used for laboratory scale expression of recombinant proteins as well as for comparing different proteins or vectors for yield or expression kinetics. The simplicity of syringe infiltration also suggests its utility in high school and college education for the subject of biotechnology. In contrast, vacuum infiltration is more robust and can be scaled-up for commercial manufacture of pharmaceutical proteins. It also offers the advantage of being able to agroinfiltrate plant species that are not amenable for syringe infiltration such as lettuce and Arabidopsis. Overall, the combination of syringe and vacuum agroinfiltration provides researchers and educators a simple, efficient, and robust methodology for transient protein expression. It will greatly facilitate the development of pharmaceutical proteins and promote science education.

Plants offer a novel system for the production of pharmaceutical proteins on a commercial scale that is more scalable, cost-efficient and safe than current expression paradigms. In this study, we report a simple and convenient, yet scalable approach to introduce target-gene containing Agrobacterium tumefaciens into plants for protein transient expression.

高级别瞬态表达重组蛋白的植物高效的农杆菌

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing ...

Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana

Agrobacterium-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short period of time. However, this technology is only just beginning to be applied to large-scale production as many technological obstacles to scale up are now being overcome. Here, we demonstrate a simple and reproducible method for industrial-scale transient protein production based on vacuum infiltration of Nicotiana plants with Agrobacteria carrying launch vectors. Optimization of Agrobacterium cultivation in AB medium allows direct dilution of the bacterial culture in Milli-Q water, simplifying the infiltration process. Among three tested species of Nicotiana, N. excelsiana (N. benthamiana &#215; N. excelsior) was selected as the most promising host due to the ease of infiltration, high level of reporter protein production, and about two-fold higher biomass production under controlled environmental conditions. Induction of Agrobacterium harboring pBID4-GFP (Tobacco mosaic virus-based) using chemicals such as acetosyringone and monosaccharide had no effect on the protein production level. Infiltrating plant under 50 to 100 mbar for 30 or 60 sec resulted in about 95% infiltration of plant leaf tissues. Infiltration with Agrobacterium laboratory strain GV3101 showed the highest protein production compared to Agrobacteria laboratory strains LBA4404 and C58C1 and wild-type Agrobacteria strains at6, at10, at77 and A4. Co-expression of a viral RNA silencing suppressor, p23 or p19, in N. benthamiana resulted in earlier accumulation and increased production (15-25%) of target protein (influenza virus hemagglutinin).

Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana

Transient protein production in Nicotiana plants based on vacuum infiltration with Agrobacteria carrying launch vectors (Tobacco mosaic virus-based) is a rapid and economic approach to produce vaccine antigens and therapeutic proteins. We simplified the procedure and improved target accumulation by optimizing conditions of bacteria cultivation, selecting host species, and co-introducing RNA silencing suppressors.

优化和利用<em&gt;农杆菌</em&gt;介导的瞬态蛋白生产中<em&gt;烟草</em

Agrobacterium-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short ...

Luciferase Complementation Imaging Assay in Nicotiana benthamiana Leaves for Transiently Determining Protein-protein Interaction Dynamics

Protein-protein interactions are fundamental mechanisms for relaying signal transduction in most cellular processes; therefore, identification of novel protein-protein interaction pairs and monitoring protein interaction dynamics are of particular interest for revealing how plants respond to environmental factors and/or developmental signals. A plethora of approaches have been developed to examine protein-protein interactions, either in vitro or in vivo. Among them, the recently established luciferase complementation imaging (LCI) assay is the simplest and fastest method for demonstrating in vivo protein-protein interactions. In this assay, protein A or protein B is fused with the amino-terminal or carboxyl-terminal half of luciferase, respectively. When protein A interacts with protein B, the two halves of luciferase will be reconstituted to form a functional and active luciferase enzyme. Luciferase activity can be recorded with a luminometer or CCD-camera. Compared with other approaches, the LCI assay shows protein-protein interactions both qualitatively and quantitatively. Agrobacterium infiltration in Nicotiana benthamiana leaves is a widely used system for transient protein expression. With the combination of LCI and transient expression, these approaches show that the physical interaction between COP1 and SPA1 was gradually reduced after jasmonate treatment.

Luciferase Complementation Imaging Assay in Nicotiana benthamiana Leaves for Transiently Determining Protein-protein Interaction Dynamics

This manuscript describes an easy and rapid experimental procedure for determining protein-protein interactions based on the measurement of luciferase activity.

荧光互补成像检测在烟草烟叶中瞬时测定蛋白质-蛋白质相互作用动力学

Protein-protein interactions are fundamental mechanisms for relaying signal transduction in most cellular processes; therefore, identification of novel ...

Production of IgG Fusion Proteins Transiently Expressed in Nicotiana benthamiana

High demand for antibodies as therapeutic interventions for various infectious, metabolic, autoimmune, neoplastic, and other diseases creates a growing need in developing efficient methods for recombinant antibody production. As of 2019, there were more than 70 FDA-approved monoclonal antibodies, and there is exponential growth potential. Despite their promise, limiting factors for widespread use are manufacturing costs and complexity. Potentially, plants offer low-cost, safe, and easily scalable protein manufacturing&#160;strategies. Plants like Nicotiana benthamiana not only can correctly fold and assemble complex mammalian proteins but also can add critical post-translational modifications similar to those offered by mammalian cell cultures. In this work, by using native GFP and an acid-stable variant of green fluorescent protein (GFP) fused to human monoclonal antibodies, we were able to visualize the entire transient antibody expression and purification process from N. benthamiana plants. Depending on the experiment&#39;s purpose, native GFP fusion can ensure easier visualization during the expression phase in the plants, while acid-stable GFP fusion allows for visualization during downstream processing. This scalable and straightforward procedure can be performed by a single researcher to produce milligram quantities of highly pure antibody or antibody fusion proteins in a matter of days using only a few small plants. Such a technique can be extended to the visualization of any type of antibody purification process and potentially many other proteins, both in plant and other expression systems. Moreover, these techniques can benefit virtual instructions and be executed in a teaching laboratory by undergraduate students possessing minimal prior experience with molecular biology techniques, providing a foundation for project-based exploration with real-world applications.

Production of IgG Fusion Proteins Transiently Expressed in Nicotiana benthamiana

We describe here a simple method for expression, extraction, and purification of recombinant human IgG fused to GFP in Nicotiana benthamiana. This protocol can be extended to purification and visualization of numerous proteins that utilize column chromatography. Moreover, the protocol is adaptable to the in-person and virtual college teaching laboratory, providing project-based exploration.

在尼科蒂亚娜本 thamiana中瞬态表达IgG融合蛋白的生产

High demand for antibodies as therapeutic interventions for various infectious, metabolic, autoimmune, neoplastic, and other diseases creates a growing ...

An Experimental Study on Colorado Potato Beetle Hibernation Under Natural Conditions

One of the major pests of potato Solanum tuberosum L. in the temperate zone is the insect Colorado potato beetle (CPB). Most studies on the immunity and diseases of the CPB are conducted during active feeding stages. Nonetheless, there are fewer studies on resting stages, although these beetles spend most of their life cycle in a state of winter diapause (hibernation). In this work, a method for investigating CPB hibernation under natural conditions was developed and tested, offering an opportunity to collect a sufficient number of individuals in winter. In this article, CPB survival was assessed, and infectious agents at different stages of hibernation were identified. CPB mortality increased during the hibernation, reaching a maximum in April-May. Entomopathogenic fungi (Beauveria, Isaria, and Lecanicillium) and bacteria Bacillus, Sphingobacterium, Peribacillus, Pseudomonas, and Serratia were isolated&#160;from the dead insects. The survival rate of the beetles for the entire hibernation period was 61%. No frozen or desiccated beetles were found, indicating the success of the presented method.

Here we present a method for studying Colorado potato beetle hibernation under the natural conditions of the temperate zone as well as a technique for collecting beetles in winter. This method allows to obtain a desired number of overwintering individuals for various analyses at any stage of hibernation.

自然条件下科罗拉多马铃薯甲虫冬眠的实验研究

One of the major pests of potato Solanum tuberosum L. in the temperate zone is the insect Colorado potato beetle (CPB). Most studies on the immunity and ...

Growing Potato Plants for Cell Death Initiation Studies

种植马铃薯植物进行细胞死亡起始研究

气候相关胁迫下的马铃薯恢复力分析

High Throughput Image-Based Phenotyping for Determining Morphological and Physiological Responses to Single and Combined Stresses in Potato

High throughput image-based phenotyping is a powerful tool to non-invasively determine the development and performance of plants under specific conditions over time. By using multiple imaging sensors, many traits of interest can be assessed, including plant biomass, photosynthetic efficiency, canopy temperature, and leaf reflectance indices. Plants are frequently exposed to multiple stresses under field conditions where severe heat waves, flooding, and drought events seriously threaten crop productivity. When stresses coincide, resulting effects on plants can be distinct due to synergistic or antagonistic interactions. To elucidate how potato plants respond to single and combined stresses that resemble naturally occurring stress scenarios, five different treatments were imposed on a selected potato cultivar (Solanum tuberosum L., cv. Lady Rosetta) at the onset of tuberization, i.e. control, drought, heat, waterlogging, and combinations of heat, drought, and waterlogging stresses. Our analysis shows that waterlogging stress had the most detrimental effect on plant performance, leading to fast and drastic physiological responses related to stomatal closure, including a reduction in the quantum yield and efficiency of photosystem II and an increase in canopy temperature and water index. Under heat and combined stress treatments, the relative growth rate was reduced in the early phase of stress. Under drought and combined stresses, plant volume and photosynthetic performance dropped with an increased temperature and stomata closure in the late phase of stress. The combination of optimized stress treatment under defined environmental conditions together with selected phenotyping protocols allowed to reveal&#160;the dynamics of morphological and physiological responses to single and combined stresses. Here, a useful tool is presented for plant researchers looking to identify plant traits indicative of resilience to several climate change-related stresses.

作者聚焦：使用 PlantScreen 机器人平台揭示植物对非生物胁迫的响应

We designed an image-based phenotyping protocol to determine the morphological and physiological responses to single and combined heat, drought, and waterlogging treatments. This approach enabled the identification of early, late, and recovery responses at a whole plant level, particularly above-ground parts, and highlighted the necessity of using multiple imaging sensors.

基于图像的高通量表型分析用于确定马铃薯对单一胁迫和联合胁迫的形态和生理响应

High throughput image-based phenotyping is a powerful tool to non-invasively determine the development and performance of plants under specific conditions ...