Tomato histone H2B monoubiquitination enzymes SlHUB1 and SlHUB2 contribute to disease resistance against Botrytis cinerea through modulating the balance between SA- and JA/ET-mediated signaling pathways

Abstract

Background: Histone H2B monoubiquitination pathway has been shown to play critical roles in regulating growth/development and stress response in Arabidopsis. In the present study, we explored the involvement of the tomato histone H2B monoubiquitination pathway in defense response against Botrytis cinerea by functional analysis of SlHUB1 and SlHUB2, orthologues of the Arabidopsis AtHUB1/AtHUB2.

Methods: We used the TRV-based gene silencing system to knockdown the expression levels of SlHUB1 or SlHUB2 in tomato plants and compared the phenotype between the silenced and the control plants after infection with B. cinerea and Pseudomonas syringae pv. tomato (Pst) DC3000. Biochemical and interaction properties of proteins were examined using in vitro histone monoubiquitination and yeast two-hybrid assays, respectively. The transcript levels of genes were analyzed by quantitative real time PCR (qRT-PCR).

Results: The tomato SlHUB1 and SlHUB2 had H2B monoubiquitination E3 ligases activity in vitro and expression of SlHUB1 and SlHUB2 was induced by infection of B. cinerea and Pst DC3000 and by treatment with salicylic acid (SA) and 1-amino cyclopropane-1-carboxylic acid (ACC). Silencing of either SlHUB1 or SlHUB2 in tomato plants showed increased susceptibility to B. cinerea, whereas silencing of SlHUB1 resulted in increased resistance against Pst DC3000. SlMED21, a Mediator complex subunit, interacted with SlHUB1 but silencing of SlMED21 did not affect the disease resistance to B. cinerea and Pst DC3000. The SlHUB1- and SlHUB2-silenced plants had thinner cell wall but increased accumulation of reactive oxygen species (ROS), increased callose deposition and exhibited altered expression of the genes involved in phenylpropanoid pathway and in ROS generation and scavenging system. Expression of genes in the SA-mediated signaling pathway was significantly upregulated, whereas expression of genes in the jasmonic acid (JA)/ethylene (ET)-mediated signaling pathway were markedly decreased in SlHUB1- and SlHUB2-silenced plants after infection of B. cinerea.

Conclusion: VIGS-based functional analyses demonstrate that both SlHUB1 and SlHUB2 contribute to resistance against B. cinerea most likely through modulating the balance between the SA- and JA/ET-mediated signaling pathways.

Fig. 1

SlHUB1 and SlHUB2 are functional histone H2B monoubiquitination E3 ligases. a Phylogenetic tree analysis of SlHUB1 and SlHUB2 with yeast BRE (GenBank accession No. Q07457), Arabidopsis AtHUB1 (Q8RXD6) and AtHUB2 (NP_564680), and human RFN20 (NP_062538) and RFN40 (NP_001273501). Sequence alignment was performed using ClustalX 1.81 program and phylogenic tree was created and visualized using MEGA 6.06. b Amino acid alignments of the SlHUB1 and SlHUB2 RING domains with RING domains of the Arabidopsis AtHUB1 and AtHUB2 and yeast BRE. Filled triangles indicate the conserved cysteine residues, while asterisk indicates conserved histidine residue. c Recombinant SlHUB1 (right) and SlHUB2 (left) proteins have histone H2B monoubiquitination activity in vitro. Recombinant SlHUB1 and SlHUB2 and their mutants SlHUB1^ΔRING and SlHUB2^ΔRING were incubated with E1 enzyme, E2 enzyme (Rad6), H2B substrate and ubiquitin, separated on SDS-PAGE and detected by Western blotting using anti-ubiquitin antibody. The absences of each one of H2B, E1, E2 or ubiquitin were included as negative controls

Fig. 2

Expression of SlHUB1 and SlHUB2 in responses to pathogens and defense signaling-related hormones. Four-week-old plants were inoculated by spore suspension of B. cinerea (a), vacuum-infiltrated by suspension of Pst DC3000 (b) or treated by foliar spraying with 1 mM SA, 100 μM MeJA, 100 μM ACC solutions or sterilized distill water as a control (c). Leaf samples were collected at indicated time points after treatment. Relative expression was shown as folds of the actin transcript values. Data presented are the means ± SD from three independent experiments and different letters above the columns indicate significant differences at p < 0.05 level

Fig. 3

Silencing of SlHUB1 and SlHUB2 resulted in reduced resistance to B. cinerea. Two-week-old seedlings were infiltrated with agrobacteria carrying pTRV2-SlHUB1, pTRV2-SlHUB2 or pTRV2-GUS constructs and disease assays were carried out at 4 weeks after VIGS infiltration. a Silencing efficiency of SlHUB1 and SlHUB2 in VIGS construct-infiltrated plants. The transcript levels of SlHUB1 or SlHUB2 in pTRV2-SlHUB1 or pTRV2-SlHUB2-infiltrated plants were analyzed by qRT-PCR and compared to that in pTRV2-GUS-infiltrated plants, which was set 1. b, d Disease phenotype (b) and lesion size (d) on detached leaves of pTRV2-SlHUB1, pTRV2-SlHUB2 or pTRV2-GUS-infiltrated plants after drop-inoculation with B. cinerea, respectively. Photographs were taken at 4 days post-inoculation (dpi). Lesion sizes were measured at 4 dpi and on a minimum of 30 leaves in each experiment. c, e Disease phenotype (c) and fungal growth (e) of pTRV2-SlHUB1, pTRV2-SlHUB2 or pTRV2-GUS-infiltrated plants after spraying with B. cinerea, respectively. Photographs were taken at 6 dpi. Growth of B. cinerea in planta was measured at 3 dpi by analyzing the transcript level of BcActinA gene with the SlActin gene as an internal control. Data presented are the means ± SD from three independent experiments and different letters above the columns indicate significant differences at p < 0.05 level

Fig. 4

SlMED21 interacts with SlHUB1 but did not affect the resistance to B. cinerea. a SlMED21 interacted with SlHUB1 in yeast two-hybrid assay. Yeasts carrying the SlMED21 in the prey vector and the SlHUB1 in the bait prey vector were assayed for growth on selective medium (SD/Leu⁻ Trp⁻ Ade⁻ His⁻) and β-galactosidase activity after addition of X-α-Gal. The positive control pGADT7-T + pGBKT7-53 and other indicated combinations between empty vector and SlHUB1/SlMED21 were assayed in parallel. b Silencing efficiency of SlMED21 in VIGS construct-infiltrated plants. The silencing efficiency was calculated by comparing the transcript levels of SlMED21 in pTRV2-SlMED21-infiltrated plants to that in pTRV2-GUS-infiltrated plants, which were set as 1. c Disease symptom on detached leaves at 3 dpi. d Disease phenotype on whole plants at 6 dpi, respectively. e Lesion sizes on selected leaves in detached leaf inoculation assays at 3 dpi. Lesion sizes were measured on a minimum of 30 leaves in each experiment. f Growth of B. cinerea in inoculated plants from the whole plant inoculation experiments at 3 dpi. Relative fungal growth was shown as folds of transcript levels of BcActin compared to SlActin. Data presented are the means ± SD from three independent experiments and different letters above the columns indicate significant differences at p < 0.05 level

Fig. 5

Silencing of SlHUB1 resulted in increased resistance to P. syringae pv. tomato DC3000. Two-week-old seedlings were infiltrated with agrobacteria carrying pTRV2-SlHUB1, pTRV2-SlHUB2, pTRV2-SlMED21 or pTRV2-GUS conducts and disease assays were performed by vacuum infiltrating with Pst DC3000 at 4 weeks after VIGS infiltration. a Representative symptom of disease caused by Pst DC3000 at 4 dpi. b Bacterial growth in inoculated leaves of pTRV2-SlHUB1-, pTRV2-SlHUB2-, pTRV2-SlMED21- or pTRV2-GUS-infiltrated plants. Leaf samples were collected at 0 and 4 days after inoculation and bacterial growth was measured. Data presented are the means ± SD from three independent experiments and different letters above the columns indicate significant differences at p < 0.05 level

Fig. 6

Silencing of SlHUB1 and SlHUB2 resulted in reduced cell wall thickness but increased callose accumulation after B. cinerea infection. Two-week-old seedlings were infiltrated with agrobacteria carrying pTRV2-SlHUB1, pTRV2-SlHUB2, pTRV2-SlMED21 or pTRV2-GUS constructs. a, b Representative TEM photos showing the cell wall (a) and the thickness of cell wall (b) in pTRV2-SlHUB1-, pTRV2-SlHUB2- or pTRV2-GUS-infiltrated plants. Leaf samples were collected for TEM assays at 4 weeks after VIGS infiltration. Bars = 200 nm. The data represent mean ± SE from 20 samples. c Callose accumulation. The VIGS construct-infiltrated plants were inoculated with B. cinerea and at least 6 leaves from 6 individual plants were collected at 0 h and 24 h after inoculation for detection of callose accumulation. Upper row represents callose staining in mock-inoculated leaves whereas lower row represents callose staining in B. cinerea-inoculated leaves. Bars = 100 μm. The callose data shown in (d) were quantified using an image analysis program as described in Methods

Fig. 7

Silencing of SlHUB1 and SlHUB2 attenuated the expression of phenylpropanoid pathway-related genes after B. cinerea infection. Two-week-old seedlings were infiltrated with agrobacteria carrying pTRV2-SlHUB1, pTRV2-SlHUB2, pTRV2-SlMED21 or pTRV2-GUS constructs and were inoculated with spore suspension of B. cinerea at 4 weeks after VIGS infiltration. At least 6 leaves from 6 individual plants were collected at 0 and 24 h after inoculation and used for analysis of gene expression. Data presented are the means ± SD from three independent experiments and different letters above the columns indicate significant differences at p < 0.05 level

Fig. 8

Silencing of SlHUB1 and SlHUB2 resulted in accumulation of ROS and affected the expression of ROS generation- and scavenging-related genes after infection with B. cinerea. Two-week-old seedlings were infiltrated with agrobacteria carrying pTRV2-SlHUB1, pTRV2-SlHUB2 or pTRV2-GUS constructs and were inoculated by spraying with spore suspension of B. cinerea or with buffer as mock-inoculation control at 4 weeks after VIGS infiltration. At least 6 leaves from 6 individual plants were collected at 0 (as controls) and 24 h after inoculation. a Accumulation superoxide anion. b Accumulation of H₂O₂. Representative NBT- or DAB-stained leaves are shown and similar results were obtained from repeated experiments. c Expression of ROS generation- and scavenging-related genes before and after infection with B. cinerea. Relative expression levels were shown as folds of the actin transcript values. Data presented are the means ± SD from three independent experiments and different letters above the columns indicate significant differences at p < 0.05 level

Fig. 9

Silencing of SlHUB1 and SlHUB2 affected the expression of SA-, JA- and ET-mediated signaling and responsive genes after Botrytis infection. Two-week-old seedlings were infiltrated with agrobacteria carrying pTRV2-SlHUB1, pTRV2-SlHUB2, pTRV2-SlMED21 or pTRV2-GUS construct and were inoculated with spore suspension of B. cinerea at 4 weeks after VIGS infiltration. At least 6 leaves from 6 individual plants were collected at 0 and 24 h after inoculation and used for analysis of gene expression. a Expression of SA-mediated signaling and responsive genes, (b) Expression of JA-mediated signaling and responsive genes and (c) Expression of ET-mediated signaling and responsive genes. Relative expression levels were shown as folds of the actin transcript values. Data presented in (b) are the means ± SD from three independent experiments and different letters above the columns indicate significant differences at p < 0.05 level