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Geminivirus C4 protein alters Arabidopsis development

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Abstract

The C4 protein of beet curly top virus [BCTV-B (US:Log:76)] induces hyperplasia in infected phloem tissue and tumorigenic growths in transgenic plants. The protein offers an excellent model for studying cell cycle control, cell differentiation, and plant development. To investigate the role of the C4 protein in plant development, transgenic Arabidopsis thaliana plants were generated in which the C4 transgene was expressed under the control of an inducible promoter. A detailed analysis of the developmental changes that occur in cotyledons and hypocotyls of seedlings expressing the C4 transgene showed extensive cell division in all tissues types examined, radically altered tissue layer organization, and the absence of a clearly defined vascular system. Induced seedlings failed to develop true leaves, lateral roots, and shoot and root apical meristems, as well as vascular tissue. Specialized epidermis structures, such as stomata and root hairs, were either absent or developmentally impaired in seedlings that expressed C4 protein. Exogenous application of brassinosteroid and abscisic acid weakly rescued the C4-induced phenotype, while induced seedlings were hypersensitive to gibberellic acid and kinetin. These results indicate that ectopic expression of the BCTV C4 protein in A. thaliana drastically alters plant development, possibly through the disruption of multiple hormonal pathways.

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Acknowledgments

The authors thank Garry Sunter (University of Texas, San Antonio, TX, USA) for providing the plasmid pUC18-BCTV dimer, Roger Innis (Indiana University, Bloomington, IN, USA) for Arabidopsis thaliana ecotype Sei-O seeds, Nam-Hai Chua for the pER10 plasmid, and Richard Meagher (University of Georgia, Athens, GA, USA) for control qRT-PCR ACT2 primers. We thank Beth Richardson and John Shields for assistance with tissue sectioning and SEM, respectively. We also thank Zheng-Hua Ye and John Sherwood for reviewing the manuscript. This work was supported by funding from the Georgia Agricultural Experiment Station.

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The authors declare that they have no conflict of interest.

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  1. Department of Plant Pathology, Plant Sciences Building, The University of Georgia, Athens, GA, 30602-7274, USA

    Katherine Mills-Lujan & Carl Michael Deom

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Correspondence to Carl Michael Deom.

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Supplemental Table 1

Primer pairs used for cloning and qRT-PCR analysis (DOC 51 kb)

Fig. S1

Expression of C4 mRNA and protein correlates with inducer concentrations and time following induction. a Northern blot analysis on total RNA extracted from seedlings germinated in liquid for 7 days prior to induction with varying concentrations of β-estradiol (0 to 10 μM) and collected 24 hpi (upper panel). Transgenic lines IPC4-28, IPC4nt-12, and IPV-20 express wild-type C4, a nontranslatable version of C4 (C4nt), and empty vector (V) from a β-estradiol inducible promoter (IP), respectively. Control lines CPC4nt-6 and CPV-25 express C4nt and empty vector from the constitutive 35S promoter (CP), respectively. rRNA loading control is indicated in the lower panel. b Western blot analysis of total protein extracted from seedlings treated as described in a. Nonspecific protein band used as loading control is indicated in the lower panel. c Western blot analysis of total protein extracted from seedlings germinated and grown in liquid for 7 days prior to induction with 10 μM β-estradiol and collected at various times after induction (upper panel). Nonspecific protein band used as loading control indicated in the lower panel. Protein molecular mass markers and C4 are indicated in b and c. β-est. β-estradiol (GIF 88 kb)

High Resolution Image (TIFF 92838 kb)

Fig. S2

In planta expression of C4 leads to loss of RAM organization. Longitudinal sections of RAMs of IPC4nt-12 seedlings germinated on media with 1.0 μM β-estradiol (A–C) and IPC4-28 seedlings germinated on media without inducer (D–F) and on media with 1.0 μM inducer (G–I), analyzed by light microscopy. Samples were taken from seedlings at 2, 3, and 8 dpg. β-est. β-estradiol, c columella; black arrowhead, quiescent center. Scale bars = 50 μm (GIF 195 kb)

High Resolution Image (TIFF 98642 kb)

Fig. S3

Quantitative real-time PCR analysis of CYCA1;1, CYCB1;4, and CDKB2;2 mitotic markers. IPC4-28 seedlings were germinated for 7 days in liquid media then treated with 0.0 or 10 μM β-estradiol. Seedlings were collected at 3, 6, 24, 48, and 96 hpi, and total RNA was extracted and assayed by qRT-PCR. The relative expression levels of each gene are indicated. Relative transcript abundance for each gene was normalized to ACT2. Data represent means ± SD for three independent biological replicates (GIF 65 kb)

High Resolution Image (TIFF 5573 kb)

Fig. S4

Effects of hormone treatments on C4-induced phenotype. Seedlings were germinated on solid media (A–D), on solid induction media (E–H), on solid induction media amended with 0.5 μM ΑΒΑ (I–L), 1 μM GA3 (M–P), or 1 μM IAA (Q–T). Induction media contained 10 μM β-est. Seedlings were observed at 6 and 15 dpg (A–P) or 6 and 12 dpg (Q–T). Arrowhead, root; β-est. β-estradiol, ABA abscisic acid, GA 3 gibberellic acid, IAA the indole-3-acetic acid. Scale bar = 1,000 μm (GIF 355 kb)

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Mills-Lujan, K., Deom, C.M. Geminivirus C4 protein alters Arabidopsis development. Protoplasma 239, 95–110 (2010). https://doi.org/10.1007/s00709-009-0086-z

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