Abstract
BackgroundTransient gene expression is a powerful tool to study gene function in plants. In citrus, Agrobacterium transformation is the method of choice for transient expression studies, but this method does not work efficiently with many gene constructs, and there is a need for a more robust transient expression system in citrus leaves. Biolistic particle delivery is an alternative to Agrobacterium transformation, and in some plants, such as Arabidopsis, gives higher transformation rates in leaf tissues than Agrobacterium.ResultsHere we describe an improved method for gene expression in epidermal cells of citrus leaves, using the Bio-Rad Helios gene-gun. Gene-gun bombardment of GFP-HDEL produced highly efficient gene expression in large number of cells and in different citrus varieties. We show here that transiently expressed proteins have maintained their functions in plants, and this is demonstrated by the subcellular localization of different organelle markers, and by a functional assay of Xanthomonas citri effector AvrGF1. To further expand the available tools for subcellular localization studies in citrus, we also generated a new set of transgenic citrus plants that contain organelle markers labelling the nuclei, actin and endoplasmic reticulum. Using these new tools, we were able to show that the coat protein of citrus tristeza virus localizes to the cytoplasm and nuclei when expressed in epidermal cells fused to GFP.ConclusionWe have optimized a new method for transient expression in citrus leaves, to give highly reproducible and efficient transformation without producing a high level of injury or artifacts to the bombarded tissue. We also generated the first set organelle markers for use in citrus. These fluorescent protein markers label the nucleus and the actin. With these new resources, protein activity and subcellular localization can be studied in citrus rapidly and in high throughput. The handheld gene-gun device can also be used in the grove to deliver therapies for citrus diseases, such as canker and Huanglongbing, into trees.
Highlights
Transient gene expression is a powerful tool to study gene function in plants
Lower gold concentration resulted in a statistically significant reduction of expression rate in Madam Vinous sweet orange (Citrus sinensis (L.) Osbeck) (Additional file 1), highlighting the level of variability found with particle bombardment between different varieties, and different bombardment events, which is a limitation of the bombardment method
We have optimized a new method for transient expression in citrus leaves, which is based on the Bio-Rad Helios gene-gun bombardment
Summary
Transient gene expression is a powerful tool to study gene function in plants. Genetic engineering is becoming widely accepted as a solution for disease control in plants, including diseases in citrus. This is especially true for the expression of plant-derived genes, and for new methods of gene editing. Agrobacterium transformation was successfully applied to various citrus cultivars, and this method has been employed to study the type III effector gene avrGf1 in grapefruit and pepper Bs2 gene in Citrus limon [5, 6]. Agrobacterium infection may carry a limitation by generating potentially unpredictable effects of bacterial effector proteins known to be exported into the plant cells together with the transforming T-DNA [10]. Adding Xcc pretreatment might even further complicate this undesirable effect, and influence the interpretation of the obtained results
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