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. 2021 Jun 4:12:684192.
doi: 10.3389/fpls.2021.684192. eCollection 2021.

Agrobacterium VirE2 Protein Modulates Plant Gene Expression and Mediates Transformation From Its Location Outside the Nucleus

Affiliations

Agrobacterium VirE2 Protein Modulates Plant Gene Expression and Mediates Transformation From Its Location Outside the Nucleus

Rachelle A Lapham et al. Front Plant Sci. .

Abstract

Agrobacterium effector protein VirE2 is important for plant transformation. VirE2 likely coats transferred DNA (T-DNA) in the plant cell and protects it from degradation. VirE2 localizes to the plant cytoplasm and interacts with several host proteins. Plant-expressed VirE2 can complement a virE2 mutant Agrobacterium strain to support transformation. We investigated whether VirE2 could facilitate transformation from a nuclear location by affixing to it a strong nuclear localization signal (NLS) sequence. Only cytoplasmic-, but not nuclear-localized, VirE2 could stimulate transformation. To investigate the ways VirE2 supports transformation, we generated transgenic Arabidopsis plants containing a virE2 gene under the control of an inducible promoter and performed RNA-seq and proteomic analyses before and after induction. Some differentially expressed plant genes were previously known to facilitate transformation. Knockout mutant lines of some other VirE2 differentially expressed genes showed altered transformation phenotypes. Levels of some proteins known to be important for transformation increased in response to VirE2 induction, but prior to or without induction of their corresponding mRNAs. Overexpression of some other genes whose proteins increased after VirE2 induction resulted in increased transformation susceptibility. We conclude that cytoplasmically localized VirE2 modulates both plant RNA and protein levels to facilitate transformation.

Keywords: Agrobacterium; Arabidopsis; VirE2; plant transformation; protein subcellular localization; proteome; transcriptome.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Subcellular localization of VirE2-Venus (A,B) and VirE2-Venus-NLS (C,D) in A. thaliana roots. Transgenic A. thaliana plants expressing inducible VirE2-Venus or VirE2-Venus-NLS were treated with β-estradiol (A,C) or control solution (B,D). Cerulean-NLS under the control of a CaMV 2 × 35S promoter was used to mark the nuclei. Root cells were imaged by confocal microscopy 9 h after treatment and representative images are shown. Four images of each cell are presented (left to right: Merged DIC + YFP + Cerulean; Cerulean; Venus; merged Venus + Cerulean). Boxes indicate an enlargement of one portion of the merged Venus + Cerulean image. Bars indicate 100 μm.
FIGURE 2
FIGURE 2
Transformation susceptibility of Arabidopsis wild-type (Col-0) and β-estradiol inducible transgenic VirE2-Venus and VirE2-Venus-NLS plants. Agrobacterium-mediated transient transformation assays were conducted on roots of three transgenic lines of inducible VirE2-Venus, three transgenic lines of inducible VirE2-Venus-NLS, and wild-type Col-0 plants. Following treatment for 24 h with β-estradiol or control solutions, root segments were inoculated with (A) 108 cfu/mL of the virE2 mutant strain A. tumefaciens At1879 containing pBISN2 or (B) 105 cfu/mL of the wild-type VirE2 strain EHA105:pBISN1 (At1529). Root segments were stained with X-gluc 6 days after infection. Bars represent an average of three biological replicates (each replicate containing >60 root segments) + SE. ANOVA test *p-value < 0.05, **p-value < 0.01, ns, not significant.
FIGURE 3
FIGURE 3
Gene Ontology (GO) Biological Process Categories of up- (A) and down-regulated (B) genes in the presence of VirE2. Displayed are categories of genes with 1.3-fold or greater change in expression, considering all time points.
FIGURE 4
FIGURE 4
Gene Ontology (GO) Enrichment Analysis of VirE2 differentially expressed genes. GO biological processes of over-represented gene categories for VirE2 differentially expressed genes at all time points. Displayed are results only with a false discovery rate (FDR) < 0.05.
FIGURE 5
FIGURE 5
Quantitative RT-PCR analysis of selected VirE2 differentially expressed genes in inducible VirE2-Venus (cytoplasmic) versus inducible VirE2-Venus-NLS (nuclear) plants. VirE2-Venus (left) and VirE2-Venus-NLS (right) results of (A) FRO2, (B) TMP, (C) HSP90, (D) LEA4-5, and (E) CBFP gene expression in induced relative to non-induced roots. Bars represent an average of three technical replicates ± SE for one representative biological replicate of one transgenic line. Relative expression is shown after 3 (LEA4-5 only) or 12 h after induction in the presence of A. tumefaciens A136.
FIGURE 6
FIGURE 6
Gene Ontology (GO) Biological Process Categories of VirE2 differentially expressed proteins. Proteins are grouped according to Gene Ontology (GO) process terms. Up-regulated proteins after 3 (A) or 12 (B) hours of VirE2 induction are shown along with down-regulated proteins after 3 (C) or 12 (D) hours of VirE2 induction. Only proteins which showed at least a 20% change in abundance for all three biological replicates determined by two different computational methods are shown. Total protein number is shown in the upper right corner of each graph and is highlighted in gray (up-regulated) or in black (down-regulated).

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