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Xcc-facilitated agroinfiltration of citrus leaves: a tool for rapid functional analysis of transgenes in citrus leaves

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Abstract

Key message

Xanthomonas citri subsp. citri pretreatment before agroinfiltration could significantly promote transient expression in citrus leaves which were previously recalcitrant to agroinfiltration.

Abstract

Transient expression via agroinfiltration is widely used in biotechnology but remains problematic in many economically important plants. Xanthomonas citri subsp. citri (Xcc)-facilitated agroinfiltration was employed to promote transient protein expression in Valencia sweet orange leaves, which are recalcitrant to agroinfiltration. However, it is unclear whether Xcc-facilitated agroinfiltration has broad application, i.e., whether Xcc-facilitated agroinfiltration could be used on other citrus varieties. In addition, we intended to investigate whether Xcc-facilitated agroinfiltration could be used to hasten transgene function assays, e.g., Cre/lox system and Cas9/sgRNA system. In this report, Xcc-facilitated agroinfiltration was further exploited to enhance β-glucuronidase (GUS) expression in five citrus varieties. Xcc-facilitated agroinfiltration also significantly increased GFP expression in six citrus varieties tested. Both GUS and GFP assays indicated that Xcc-facilitated agroinfiltration had the best performance in grapefruit. After Xcc-facilitated agroinfiltration was carried out in grapefruit, protoplast analysis of the transformed cells indicated that there were more than 20 % leaf cells expressing GFP. In grapefruit, usefulness of Xcc-facilitated agroinfiltration was assayed in three case studies: (1) fast functional analysis of Cre/lox system, (2) the heat shock regulation of HSP70B promoter derived from Arabidopsis, and (3) Cas9/sgRNA-mediated genome modification.

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Abbreviations

AtHSP70BP:

The Arabidopsis heat shock-regulated protein 70B promoter

CaMV 35S:

The cauliflower mosaic virus 35S promoter

GFP:

Green fluorescent protein

GUS:

β-Glucuronidase

NosT:

The nopaline synthase gene terminator

PDS:

Phytoene desaturase

35T:

The cauliflower mosaic virus 35S terminator

Xcc:

Xanthomonas citri subsp. citri

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Acknowledgments

We thank Prof. Rongxiang Fang for kindly providing pGNG and pCambia1300-Cre. We thank Lisa Malyak for critical reading of this manuscript. This research has been supported by grant from the Citrus Research and Development Foundation.

Conflict of interest

The authors declare that they have no conflict of interest.

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Authors and Affiliations

  1. Citrus Research and Education Center, Department of Microbiology and Cell Science, University of Florida, Lake Alfred, Fl, USA

    Hongge Jia & Nian Wang

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  1. Hongge Jia
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  2. Nian Wang
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Correspondence to Nian Wang.

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Communicated by Leandro Peña.

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Jia, H., Wang, N. Xcc-facilitated agroinfiltration of citrus leaves: a tool for rapid functional analysis of transgenes in citrus leaves. Plant Cell Rep 33, 1993–2001 (2014). https://doi.org/10.1007/s00299-014-1673-9

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  • DOI: https://doi.org/10.1007/s00299-014-1673-9

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