Disrupting the Homeostasis of High Mobility Group Protein Promotes the Systemic Movement of Bamboo mosaic virus

Abstract

Viruses hijack various organelles and machineries for their replication and movement. Ever more lines of evidence indicate that specific nuclear factors are involved in systemic trafficking of several viruses. However, how such factors regulate viral systemic movement remains unclear. Here, we identify a novel role for Nicotiana benthamiana high mobility group nucleoprotein (NbHMG1/2a) in virus movement. Although infection of N. benthamiana with Bamboo mosaic virus (BaMV) decreased NbHMG1/2a expression levels, nuclear-localized NbHMG1/2a protein was shuttled out of the nucleus into cytoplasm upon BaMV infection. NbHMG1/2a knockdown or even overexpression did not affect BaMV accumulation in inoculated leaves, but it did enhance systemic movement of the virus. Interestingly, the positive regulator Rap-GTPase activation protein 1 was highly upregulated upon infection with BaMV, whereas the negative regulator thioredoxin h protein was greatly reduced, no matter if NbHMG1a/2a was silenced or overexpressed. Our findings indicate that NbHMG1/2a may have a role in plant defense responses. Once its homeostasis is disrupted, expression of relevant host factors may be perturbed that, in turn, facilitates BaMV systemic movement.

Keywords: Bamboo mosaic virus; high mobility group proteins; nucleoprotein; plant-virus interaction; virus movement.

FIGURE 1

Phylogenetic and expression analyses of Nicotiana benthamiana HMG1/2 genes. (A) Similarities of NbHMG accessions to Arabidopsis thaliana HMGB3. Two NbHMG accessions with high similarity to AtHMGB3 were obtained by blasting the AtHMGB3 coding sequence against the N. benthamiana database (Sol Genomics Networks), i.e., NbHMG_101Scf02041g04034 (NbHMG1/2a) and NbHMG_101Scf09442g05005 (NbHMG1/2b). (B) Phylogenetic analysis of NbHMG1/2 with orthologs from other species. The HMG phylogeny was generated using the Neighbor Joining method in MEGA 6.0 software. Numbers represent relative phylogenetic distance. BaMV capsid protein (CP) was used as an outgroup to root the tree. (C) Relative expression levels of NbHMG1/2a by RTqPCR in N. benthamiana plants infected with BaMV infectious clone (pKBG) compared with plants infiltrated with empty vector (pKn). Inoculated leaves were tested at 3 days post infiltration (dpi), and the systemic leaves were tested at 6 dpi. Actin was used as an internal control. Data represent the mean ± standard deviation from three biological replicates. One-sided student t-tests were performed to determine significant differences. Asterisks indicate significant differences relative to control lines (infiltrated with pKn), with ** representing P < 0.01.

FIGURE 2

Domain analysis and localization of NbHMG1/2a. (A) Predicted domains encoded in NbHMG1/2a: a nuclear localization signal (NLS) between 4 and 56 amino acids (aa), a nucleolar localization signal (NoLS) between 15 and 42 aa, and a DNA-binding domain between 36 and 101 aa. No nuclear export signal was found. (B) NbHMG1/2a localizes in the nucleus. N. benthamina leaves were infiltrated with Agrobacterium carrying pBin61-HA-HMG-mCherry or with control vector pBin61-HA-mCherry. (C) NbHMG1/2a localizes in the nucleolus. N. benthamina leaves were infiltrated with Agrobacterium carrying HA-mCherry and nucleolus-localized fibrillarin fused with eGFP (eGFP-Fib), or with HA-mCherry-HMG and eGFP-Fib. DAPI was used for nuclear staining. Images were taken 3 days after infiltration, and bars represent a measurement scale of 20 and 10 μm for the nucleus and nucleolus, respectively. The experiment was repeated three times with similar results and representative images are shown.

FIGURE 3

Effects of silencing NbHMG1/2a on plant growth and resistance to BaMV. (A) Schematic illustration of our VIGS experimental design. The first three leaves of N. benthamiana plants were infiltrated with the silencing vector TRV carrying the NbHMG1/2a fragment or control mCherry fragment. Eight days later, leaves 7 and 8 were infiltrated with pBin61 carrying BaMV (pKBG). Inoculated leaves (leaves 7 and 8) and systemic leaves (leaves 11 and 12) were collected at 3, 4, and 6 days after BaMV infection (dpi). (B) Phenotype of NbHMG1/2a-silenced plants. Plants infiltrated with TRV-mCherry, TRV-HMG, and TRV-PDS. Photos were taken 8 days after agroninfiltration with TRVs. Upper and middle panel show plant height and spread, and lower panel shows leaf size. (C,D) RNA blot of BaMV in the inoculated leaves (C) and systemic leaves (D) of control plants (infiltrated with TRV-mCherry) and NbHMG1/2a-silenced plants (HMG-VIGS). The second panel represents rRNA levels as internal control. The third panel shows NbHMG1/2a levels in the control and silenced plants. eIF1α was used as an internal control. Values represent average accumulation of BaMV genomic RNA from three biological replicates ± standard deviation. Lower panels represent protein blots of BaMV capsid protein (CP) (25 kDa) and actin in the control and silenced plants.

FIGURE 4

Overexpression of NbHMG1/2a enhances systemic movement of BaMV. (A) Twenty-four-day-old N. benthamiana plants were co-agroinfiltrated with pKBG and pBin-HA-mCherry or pBin-HA-mCherry-HMG. Samples were collected from inoculated and systemic leaves at 4, 5, and 6 dpi. Upper panels are Northern blots for BaMV, with rRNA used as a loading control. Lower panels are protein blots of BaMV CP and HA-tag to detect HA-mCherry (30 kDa) and HA-mCherry-HMG (45 kDa). Ponceau S was used as a loading control. The experiment was conducted with three biological replicates, which generated similar results. Values below Northern blots represent average accumulation of BaMV genomic RNA from three biological replicates ± SD. (B) Relative expression of endogenous NbHMG1/2a and the overexpressed gene NbHMG1/2a-mCherry in inoculated and systemic leaves of NbHMG1/2a-overexpressing plants based on RTqPCR. According to one-sided student t-test, asterisk indicates significant difference for the expression of NbHMG1/2a-mCherry relative to the endogenous NbHMG1/2a, with ** representing P < 0.01. Actin was used as an internal control. Error bars represent the standard deviation from three biological replicates.

FIGURE 5

Relative expression of host genes involved in BaMV movement in NbHMG1/2a- knockdown and -overexpressing plants. Transcript levels of RAB-GAP1 (A,E), CK2α (B,F), STKL (C,G), and TRXh2 (D,H) in N. benthamiana plants infected with pKBG or pKn and upon HMG being knocked-down or overexpressed, respectively. Expression level was measured in the BaMV-inoculated leaves at 6 dpi in both HMG-silenced and HMG-overexpressed plants. Actin was used as an internal control. Data represent the mean ± SD from three biological replicates. One-sided Student’s t-tests were performed to determine significant differences. Asterisks indicate significant differences relative to mock lines (infiltrated with pKn and pBin-mCherry), with * representing P < 0.05.

FIGURE 6

Localization of NbHMG1/2a in BaMV-GFP-infected N. benthamiana. (A) Leaves of N. benthamiana were agroinfiltrated with pKBG (BaMV-GFP) (OD = 0.005) into the region represented by the green circle. Five days later, inoculated leaves were agroinfiltrated with mCherry-HMG (OD = 0.5) in whole leaves. Samples were excised (white circles) 2 days afterward for confocal microscopic observation. To detect the nucleus, 4′,6-diamidino-2-phenylindole (DAPI) solution was infiltrated into the N. benthamiana leaves before observation. Scale bars are 50 μm. Arrows and arrowheads indicate nucleus and cytoplasm, respectively. The experiment was repeated three times with similar results, and representative images are shown. (B) Protein gel blot analysis of HA-mCherry and HA-mCherry-HMG at 2 dpi by anti-HA or anti-actin body. (C) Protein gel blot analysis of HA-mCherry-HMG in subcellular fractions of BaMV infected leaf tissues. Histone H2 was used as a nuclear marker, and cytosolic tubulin served as a cytosolic marker. N, nuclear protein extracts; S, total protein extracts depleted of nuclei.