MOA Key Lab of Pest Monitoring and Green Management, College of Plant Protection, China Agricultural University, Beijing 100193, China.
Int J Mol Sci. 2023 Apr 21;24(8):7636. doi: 10.3390/ijms24087636.
-mediated transient expression (AMTE) has been widely used for high-throughput assays of gene function in diverse plant species. However, its application in monocots is still limited due to low expression efficiency. Here, by using histochemical staining and a quantitative fluorescence assay of β-glucuronidase (GUS) gene expression, we investigated factors affecting the efficiency of AMTE on intact barley plants. We found prominent variation in GUS expression levels across diverse vectors commonly used for stable transformation and that the vector pCBEP produced the highest expression. Additionally, concurrent treatments of plants with one day of high humidity and two days of darkness following agro-infiltration also significantly increased GUS expression efficiency. We thus established an optimized method for efficient AMTE on barley and further demonstrated its efficiency on wheat and rice plants. We showed that this approach could produce enough proteins suitable for split-luciferase assays of protein-protein interactions on barley leaves. Moreover, we incorporated the AMTE protocol into the functional dissection of a complex biological process such as plant disease. Based on our previous research, we used the pCBEP vector to construct a full-length cDNA library of genes upregulated during the early stage of rice blast disease. A subsequent screen of the library by AMTE identified 15 candidate genes (out of ~2000 clones) promoting blast disease on barley plants. Four identified genes encode chloroplast-related proteins: OsNYC3, OsNUDX21, OsMRS2-9, and OsAk2. These genes were induced during rice blast disease; however, constitutive overexpression of these genes conferred enhanced disease susceptibility to in . These observations highlight the power of the optimized AMTE approach on monocots as an effective tool for facilitating functional assays of genes mediating complex processes such as plant-microbe interactions.
介导的瞬时表达（AMTE）已被广泛用于不同植物物种中基因功能的高通量测定。然而，由于表达效率低，其在单子叶植物中的应用仍然有限。在这里，通过使用组织化学染色和β-葡萄糖醛酸酶（GUS）基因表达的定量荧光测定，我们研究了影响 AMTE 在完整大麦植株上效率的因素。我们发现，在常用于稳定转化的各种载体中，GUS 表达水平存在显著差异，并且载体 pCBEP 产生的表达最高。此外，在农杆菌浸润后紧接着进行一天的高湿度和两天的黑暗处理，也显著提高了 GUS 表达效率。因此，我们建立了一种优化的大麦上高效 AMTE 方法，并进一步证明了其在小麦和水稻植株上的效率。我们表明，该方法可以在大麦叶片上产生足够的蛋白质，适合用于蛋白质-蛋白质相互作用的分裂萤光素酶测定。此外，我们将 AMTE 方案纳入了植物疾病等复杂生物学过程的功能解析中。基于我们之前的研究，我们使用 pCBEP 载体构建了一个在水稻稻瘟病早期上调的全长 cDNA 文库。随后通过 AMTE 对文库进行筛选，在大麦植株上鉴定出 15 个候选基因（在约 2000 个克隆中）促进了稻瘟病。鉴定出的 4 个基因编码与叶绿体相关的蛋白质：OsNYC3、OsNUDX21、OsMRS2-9 和 OsAk2。这些基因在水稻稻瘟病中被诱导；然而，这些基因的组成型过表达导致对 的易感性增强。这些观察结果突出了优化后的 AMTE 方法在单子叶植物中的强大功能，作为一种有效的工具，可促进介导复杂过程（如植物-微生物相互作用）的基因功能测定。
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