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Huazhong Agricultural University

7 ARTICLES PUBLISHED IN JoVE

Biology

Agroinfiltration and PVX Agroinfection in Potato and Nicotiana benthamiana

Juan Du^1,2, Hendrik Rietman¹, Vivianne G. A. A. Vleeshouwers¹

¹Wageningen UR Plant Breeding, Wageningen University, ²Key Laboratory of Horticultural Plant Biology, Huazhong Agricultural University, Ministry of Education, National Centre for Vegetable Improvement (Central China), Potato Engineering and Technology Research Centre of Hubei Province, Huazhong Agricultural University

Agroinfiltration and PVX agroinfection are routine functional assays for transient ectopic expression of genes in plants. These methods are efficient assays in effectoromics strategies (rapid resistance and avirulence gene discovery) and crucial to modern research in molecular plant pathology. They meet the demand for robust high-throughput functional analysis in plants.

Genetics

QTL Mapping and CRISPR/Cas9 Editing to Identify a Drug Resistance Gene in Toxoplasma gondii

Bang Shen¹, Robin H. Powell², Michael S. Behnke²

¹State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, ²Pathobiological Sciences, School of Veterinary Medicine, Louisiana State University

Details are presented on how QTL mapping with a whole genome sequence based genetic map can be used to identify a drug resistance gene in Toxoplasma gondii and how this can be verified with the CRISPR/Cas9 system that efficiently edits a genomic target, in this case the drug resistance gene.

Cancer Research

Detection of Mitochondria Membrane Potential to Study CLIC4 Knockdown-induced HN4 Cell Apoptosis In Vitro

Jinsen Lu¹, Lele Wu², Xiaoke Wang², Jinhang Zhu², Juan Du², Bing Shen²

¹School of Basic Medical Sciences, Anhui Medical University, ²Department of Orthopedics, Anhui Provincial Hospital, Anhui Medical University

Here we present a detailed protocol for the application of rhodamine 123 to identify the mitochondrial membrane potential (MMP) and study CLIC4 knockdown-induced HN4 cell apoptosis in vitro. Under common fluorescence microscope and confocal laser scanning fluorescence microscope, the real-time change of the MMP was recorded.

Biochemistry

Expression and Purification of the Human Lipid-sensitive Cation Channel TRPC3 for Structural Determination by Single-particle Cryo-electron Microscopy

Emery Haley^*1, Wooyoung Choi^*1, Chen Fan¹, Weinan Sun^2,3, Juan Du¹, Wei Lü¹

¹Van Andel Institute, ²Vollum Institute, ³Janelia Research Campus

This protocol describes techniques used to determine ion channel structures by cryo-electron microscopy, including a baculovirus system used to efficiently express genes in mammalian cells with minimum effort and toxicity, protein extraction, purification, and quality checking, sample grid preparation and screening, as well as data collection and processing.

Biology

Determination of Self-(In)compatibility and Inter-(In)compatibility Relationships in Citrus Using Manual Pollination, Microscopy, and S-Genotype Analyses

Muhammad Husnain Ahmad¹, Xin Zheng¹, Yu Hu¹, Huimin Liu¹, Yi Sun¹, Hao Wen¹, Lijun Chai¹

¹National Key Laboratory for Germplasm Innovation & Utilization of Horticultural Crops, Huazhong Agricultural University

This protocol provides a rapid method for determining pollen compatibility and incompatibility in citrus cultivars.

Environment

Measuring Bacterial Colonization on Arabidopsis thaliana Roots in Hydroponic Condition

Xia Shu^1,2, Huatai Li¹, Jing Wang³, Shan Wang⁴, Yunpeng Liu¹, Ruifu Zhang^1,3

¹State Key Laboratory of Efficient Utilization of Arid and Semi-arid Arable Land in Northern China, Institute of Agricultural Resources and Regional Planning, Chinese Academy of Agricultural Sciences, ²State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, ³Jiangsu Provincial Key Lab for Organic Solid Waste Utilization, National Engineering Research Center for Organic-based Fertilizers, Jiangsu Collaborative Innovation Center for Solid Organic Waste Resource Utilization, Nanjing Agricultural University, ⁴Jiangsu Engineering and Technology Center for Modern Horticulture, Jiangsu Vocational College of Agriculture and Forestry

Colonization of plant growth-promoting rhizobacteria (PGPR) in the rhizosphere is essential for its growth-promoting effect. It is necessary to standardize the method of detection of bacterial rhizosphere colonization. Here, we describe a reproducible method for quantifying bacterial colonization on the root surface.

Biology

An Approach to Constructing Multispecies Biofilm Communities from Rhizosphere Soil

Pan Wang¹, Bin Xia¹, Zhiyuan Chen¹, Jiyu Xie¹, Xinli Sun¹, Qingyun Tian², Chun-hui Gao², Weibin Xun¹, Nan Zhang¹, Ruifu Zhang¹, Qirong Shen¹, Zhihui Xu¹

¹Jiangsu Provincial Key Lab for Solid Organic Waste Utilization, Key lab of organic-based fertilizers of China, Jiangsu Collaborative Innovation Center for Solid Organic Wastes, Educational Ministry Engineering Center of Resource-saving fertilizers, Nanjing Agricultural University, ²National Key Laboratory of Agricultural Microbiology and College of Resources and Environment, Huazhong Agricultural University

A rapid and standardized procedure for establishing synergistic multispecies biofilm communities from various rhizosphere soils is presented here. It is a unique protocol designed to probe and simulate the complex rhizosphere soil microbiota.