Rice is a vital food source, providing nourishment for more than 50% of the world's population. However, this crop plant, like other cultivated plants, is vulnerable to adverse environmental factors such as abiotic and biotic stresses that occasionally and severely affect rice production and yield. When rice is stressed, self-defense mechanisms, including expression of genes, proteins, and metabolites used to lessen the effects of the particular stress, are activated. Among these molecular responses, proteins constitute a critical and functional line of defense toward protecting rice plant growth and development. The role of these proteins, including proteins secreted into the extracellular environment, is of great interest to biologists focused on understanding stress responses.
In this context, transient protein expression is a useful technique for investigating protein localization and functional analysis of stress responses in rice plants. Currently, the most widely-used in planta transient protein expression techniques use three different methods, including Agrobacterium-mediated transformation, protoplast transformation using polyethylene glycol (PEG) or electroporation, and biolistic bombardment expression 1-5. Agrobacterium-mediated transformation has been well established using various rice tissues, such as shoot apex, embryonic callus, and scutellum tissue2,3. However, the production of transgenic plants takes approximately 3 to 4 months, a significant amount of time in the fast-paced research environment. In the case of PEG or electroporation-mediated transformation, protoplast tissues have been widely used5. This method is advantageous in that it is rapid. However, the loss of cell wall structure makes it difficult to detect proteins accumulating outside of the membrane. For this reason, a new biolistic bombardment method was established in which high-pressure gas is used to deliver foreign DNA into cells. Biolistic bombardment is a time-honored technique that has been used with many plants, and provides an efficient method for subcellular localization, cell-to-cell movement, and protein-protein interaction studies6-9. However, biolistic bombardment is not well suited for rice, as the leaf epidermis consists of irregular cell layers and produces strong auto-fluorescence10. Nevertheless, biolistic bombardment has been used for generating transgenic plants and for confirming protein secretion, but it has not been used for transient protein expression studies to date7-9.
We developed a protocol that uses biolistic bombardment to analyze gene expression and subcellular localization in rice. Evenly-sliced leaf sheath tissue, a clear cell structure with little auto-fluorescence, was used for the biolistic bombardment assay. Expression efficiencies of nearly 30%, which were similar to levels in previously-published Arabidopsis and rice callus systems6,11, were observed using a mCherry reporter gene expressed under the maize ubiquitin promoter. Our protocol provides a rapid and efficient way to analyze expression and subcellular localization of proteins of interest in rice cells.
This biolistic bombardment protocol is also an appropriate tool for analyzing protein secretion. Protein secretion is an important mechanism for plant growth and regulation under normal and abnormal (abiotic and biotic stress) environmental conditions12-17. For example, during the very early time periods of host-pathogen interactions when penetration by the invader occurs, both the host and the pathogen secrete large amounts of proteins into the host’s apoplastic region, a space between the cell wall and the plasma membrane18-22. The secreted proteins from the host and pathogen interact with each other in this apoplastic region23. Thus, the analysis of protein secretion and protein interactions between the host and pathogen proteins in the extracellular space is critical to understanding plant-pathogen interactions. To study the functions of pathogen-secreted effectors, transgenic Magnaporthe oryzae (rice blast fungus) mutants carrying target genes fused with fluorescent reporters have been generated and expressed in planta21,24,25. However, functional analysis of secreted effectors is difficult due to the low-efficiency and time-consuming process of fungal mutant generation. Therefore, the application of transient expression is sufficient for the study on plant-microbe interaction.
In our study, secreted proteins were analyzed in rice leaf sheaths using florescent reporters. Proteins localized outside the membrane were strongly expressed, suggesting that our protocol is suitable for in planta secretion analyses in rice. Effector proteins fused with fluorescent reporters were directly expressed in host cells. The interactions between host and pathogen proteins were observed within 24-48 h after bombardment. Therefore, our protocol provides a direct in planta expression method for rice that reduces the number of steps necessary for fungal mutant generation for analyzing the fungal secretome. A successful activation of defense reporter has been reported after transient expression of M. oryzae secretory proteins in rice sheath cells, suggesting this protocol makes possible large screens of pathogen effectors in rice26,27.
To demonstrate the further utility and specificity of our approach, we compared the localization of the secreted proteins in rice with their localization in commonly studied onion epidermis. Differential expression of the two proteins in the rice and onion systems suggested that protein secretion mechanisms vary between host and non-host plants. Therefore, setting up a rice transient expression system is essential for in planta functional analyses in rice.
Advantages and limitations of the protocol
This protocol can be widely applied for protein expression, subcellular localization, and protein-protein interaction analyses. Rice transient expression is also a valuable tool for rice-pathogen interaction research. Furthermore, this protocol can be adapted for transient gene expression in other monocotyledon plants with minor modifications for sheath preparation, particle delivery pressure, or incubation times for signal detection.
This protocol clearly describes a rapid method for developing rice in planta transient expression and protein secretion. However, a few disadvantages still remain in the protocol. Setting up a biolistic particle delivery system and confocal fluorescent microscopy can be costly. As with other techniques for the study of rice, nearly 5 weeks is required to grow the rice for sheath sample preparation prior to the experiment. As preparation of the sheaths and sectioning can be difficult, practice is required.
The following points should be considered before beginning an experiment.
Selection of microcarrier. Tungsten M-17 particles (1.1 μm) were used for gene delivery in our protocol rather than gold particles. Tungsten particles are approximately the same size as gold particles, but the surfaces are a bit rougher compared with that of gold particles. Even so, tungsten particles still showed high delivery efficiencies in our experiments with reduced experimental cost.
Rice and sheath preparation. Rice plants at the four- to six-leaf stage were grown in a greenhouse and used for sheath preparation. At this stage, the sheath tissue has less lignifications, but a sufficient thickness for slicing. Furthermore, biochemical activities are functional to express genes transformed at this age.
Set of controls. A positive control to confirm the efficiency of transient expression should always be included in the experimental design. Fluorescent reporter genes under a constitutively-expressing promoter can be used for the protein subcellular localization analyses, and any rice signal peptides, such as glucanase, can be used as protein-secretion controls. Transgenic plants carrying GFP or YFP reporters can be used as positive controls in which the target protein and mCherry reporters could be co-expressed.