Hijacking of the Host SCF Ubiquitin Ligase Machinery by Plant Pathogens

The SCF (SKP1-CUL1-F-box protein) ubiquitin ligase complex mediates polyubiquitination of proteins targeted for degradation, thereby controlling a plethora of biological processes in eukaryotic cells. Although this ubiquitination machinery is found and functional only in eukaryotes, many non-eukaryotic pathogens also encode F-box proteins, the critical subunits of the SCF complex. Increasing evidence indicates that such non-eukaryotic F-box proteins play an essential role in subverting or exploiting the host ubiquitin/proteasome system for efficient pathogen infection. A recent bioinformatic analysis has identified more than 70 F-box proteins in 22 different bacterial species, suggesting that use of pathogen-encoded F-box effectors in the host cell may be a widespread infection strategy. In this review, we focus on plant pathogen-encoded F-box effectors, such as VirF of Agrobacterium tumefaciens, GALAs of Ralstonia solanacearum, and P0 of Poleroviruses, and discuss the molecular mechanism by which plant pathogens use these factors to manipulate the host cell for their own benefit

Interaction of Arabidopsis Trihelix-Domain Transcription Factors VFP3 and VFP5 with Agrobacterium Virulence Protein VirF.

This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.Agrobacterium is a natural genetic engineer of plants that exports several virulence proteins into host cells in order to take advantage of the cell machinery to facilitate transformation and support bacterial growth. One of these effectors is the F-box protein VirF, which pre- sumably uses the host ubiquitin/proteasome system (UPS) to uncoat the packaging pro- teins from the invading bacterial T-DNA. By analogy to several other bacterial effectors, VirF most likely has several functions in the host cell and, therefore, several interacting part- ners among host proteins. Here we identify one such interactor, an Arabidopsis trihelix- domain transcription factor VFP3, and further show that its very close homolog VFP5 also interacted with VirF. Interestingly, interactions of VirF with either VFP3 or VFP5 did not acti- vate the host UPS, suggesting that VirF might play other UPS-independent roles in bacterial infection. To better understand the potential scope of VFP3 function, we used RNAi to reduce expression of the VFP3 gene. Transcriptome profiling of these VFP3-silenced plants using high-throughput cDNA sequencing (RNA-seq) revealed that VFP3 substantially affected plant gene expression; specifically, 1,118 genes representing approximately 5% of all expressed genes were significantly either up- or down-regulated in the VFP3 RNAi line compared to wild-type Col-0 plants. Among the 507 up-regulated genes were genes impli- cated in the regulation of transcription, protein degradation, calcium signaling, and hormone metabolism, whereas the 611 down-regulated genes included those involved in redox regu- lation, light reactions of photosynthesis, and metabolism of lipids, amino acids, and cell wall. Overall, this pattern of changes in gene expression is characteristic of plants under stress. Thus, VFP3 likely plays an important role in controlling plant homeostasis.Peer reviewedFinal Published versio

VFP3 interacts with VirF in the cell nucleus.

<p>(A-C) BiFC assay for the VFP3-VirF interaction in planta. Constructs encoding nYFP-VFP3 and cYFP-VirF were coexpressed in microbombarded <i>N</i>. <i>benthamiana</i> leaves. (A) Plastid autofluorescence. (B) YFP signal. (C) Merged plastid autofluorescence and YFP signals. (D-F) BiFC assay for the VFP3-VBF interaction in planta. Constructs encoding nYFP-VFP3 and cYFP-VBF were coexpressed in microbombarded <i>N</i>. <i>benthamiana</i> leaves. (D) Plastid autofluorescence. (E) YFP signal. (F) Merged plastid autofluorescence and YFP signals. (G-I) Subcellular localization of CFP-tagged VFP3 coexpressed with free DsRed2 in agroinfiltrated <i>N</i>. <i>benthamiana</i> leaves. (G) DsRed2 signal. (H) CFP signal. (I) Merged plastid autofluorescence, DsRed2 and CFP signals. Location of the cell nucleus is indicated by a white arrowhead. All images are projections of single confocal sections. Scale bars, 20 μm.</p

Percentage distribution of up- and down-regulated genes in <i>VFP3</i> RNAi-1 plants as compared to the wild-type Col-0 plants.

<p>Annotation is based on MapMan categories. Categories with gene number less than 10 are not shown. Gray bars indicate up-regulated gene categories, and blue bars indicate down-regulated gene categories.</p

Amino acid sequence analysis of VFP3.

<p>(A) Sequence alignment of VFP3 and its homologs from Arabidopsis. The deduced amino acid sequence of VFP3 (At3g11100) was aligned with the sequences of proteins encoded by At5g05550 (VFP5) and At3g58630 of Arabidopsis and of tobacco NtSIP1 (GenBank accession number BAB83610.1) using ClustalX (ver. 2.1) (<a href="http://www.clustal.org/clustal2/" target="_blank">http://www.clustal.org/clustal2/</a>). Three α-helices of the trihelix domain, delineated with an open box, and the fourth C-terminal α-helical region were predicted using the Garnier-Robson-Osguthorpe (GOR) algorithm [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142128#pone.0142128.ref026" target="_blank">26</a>]. The MADF domain, predicted by InterPro (<a href="http://www.ebi.ac.uk/interpro" target="_blank">http://www.ebi.ac.uk/interpro</a>), is delineated with a gray box. Asterisks indicate the putative monopartite NLS predicted by cNLS Mapper (nls-mapper.iab.keio.ac.jp). Identical residues in the aligned sequences are highlighted in white letters on black/dark gray background and similar residues are shaded in gray. (B) Ribbon diagram of the trihelix domain VFP3 showing the three predicted helical structures was constructed using the Hhpred (<a href="http://toolkit.tuebingen.mpg.de/" target="_blank">http://toolkit.tuebingen.mpg.de/</a>) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142128#pone.0142128.ref059" target="_blank">59</a>] and UCSF Chimera tools [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142128#pone.0142128.ref060" target="_blank">60</a>]. (C) Phylogenetic tree of the members of the SIP1 clade of Arabidopsis trihelix transcription factors and tobacco NtSIP1. VFP3 (At3g11100) and its close homolog VFP5 (At5g05550) are highlighted by a shaded box and white letters. Known gene names are indicated in parenthesis next to their locus names. The evolutionary history was inferred using the Neighbor-Joining method [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142128#pone.0142128.ref061" target="_blank">61</a>]. The optimal tree with the sum of branch length of 6.60887794 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1,000 replicates) are shown next to the branches [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142128#pone.0142128.ref062" target="_blank">62</a>]. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142128#pone.0142128.ref063" target="_blank">63</a>] and are in the units of the number of amino acid substitutions per site. All positions containing gaps and missing data were eliminated. There were a total of 199 positions in the final dataset. Evolutionary analyses were conducted using the Molecular Evolutionary Genetics Analysis tool (MEGA, version 6.0.5 for Mac OS) (<a href="http://www.megasoftware.net" target="_blank">http://www.megasoftware.net</a>) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142128#pone.0142128.ref064" target="_blank">64</a>], which also generated this description of the analysis. Bar, 0.2 amino acid substitutions per site.</p

Reduction of <i>VFP5</i> gene expression in <i>VFP3</i> RNAi-1 plants.

<p>(A) Semi-quantitative RT-PCR analysis of the <i>VFP5</i> transcript in leaves of the wild-type Col-0 and <i>VFP3</i> RNAi-1 plants. <i>ACTIN2</i> was used as internal reference. (B) Quantification of <i>VFP5</i> transcript levels described in (A) normalized to the levels of the <i>ACTIN2</i> reference. The data represent average values of three independent experiments with indicated standard deviations.</p

Metabolism overview of genes differentially expressed in <i>VFP3</i> RNAi-1 plants.

<p>The analysis employed the MapMan software. Values are log2 fold changes between the analyzed plants. Blue indicates up-regulation in gene expression, and red indicates down-regulation in gene expression.</p

Suppression of <i>VFP3</i> gene expression in <i>VFP3</i> RNAi-1 plants has no detectable effect on their genetic transformation by Agrobacterium.

<p>Root explants were infected with Agrobacterium cultures at the indicated optical densities. (A) Numbers of tumors and roots scored for each plant. (B) Tumorigenicity expressed as percent of roots showing tumors. Black bars, wild-type plants; gray bars, <i>VFP3</i> RNAi-1 plants. Standard deviations are indicated.</p

Reduction of <i>VFP3</i> gene expression in <i>VFP3</i> RNAi-1 plants.

<p>(A) Semi-quantitative RT-PCR analysis of the <i>VFP3</i> transcript levels in leaves of the wild-type Col-0 and <i>VFP3</i> RNAi-1 plants. <i>ACTIN2</i> was used as internal reference. (B) Quantification of <i>VFP3</i> transcript levels described in (A) normalized to the levels of the <i>ACTIN2</i> reference. (C) Semi-quantitative RT-PCR analysis of the <i>VFP3</i> transcript in roots of the wild-type Col-0 and <i>VFP3</i> RNAi-1 plants. <i>ACTIN2</i> was used as internal reference. (D) Quantification of <i>VFP3</i> transcript levels described in (C) normalized to the levels of the <i>ACTIN2</i> reference. The data represent average values of three independent experiments with indicated standard deviations.</p