HISTONE MONOUBIQUITINATION1 interacts with a subunit of the mediator complex and regulates defense against necrotrophic fungal pathogens in Arabidopsis

Abstract

This work examines the role of the Arabidopsis thaliana RING E3 ligase, HISTONE MONOUBIQUITINATION1 (HUB1) in disease resistance. Loss-of-function alleles of HUB1 show increased susceptibility to the necrotrophic fungal pathogens Botrytis cinerea and Alternaria brassicicola, whereas HUB1 overexpression conferred resistance to B. cinerea. By contrast, responses to the bacterial pathogen Pseudomonas syringae are unaltered in hub1 plants. hub1 mutants have thinner cell walls but increased callose around an infection site. HUB1 acts independently of jasmonate, but ethylene (ET) responses and salicylate modulate the resistance of hub1 mutants to necrotrophic fungi. The ET response factor ETHYLENE INSENSITIVE2 is epistatic to HUB1 for A. brassicicola resistance but additive to HUB1 for B. cinerea resistance. HUB1 interacts with MED21, a subunit of the Arabidopsis Mediator, a conserved complex that regulates RNA polymerase II. RNA interference lines with reduced MED21 expression are highly susceptible to A. brassicicola and B. cinerea, whereas T-DNA insertion alleles are embryonic lethal, suggesting an essential role for MED21. However, HUB1-mediated histone H2B modification is independent of histone H3 and DNA methylation. In sum, histone H2B monoubiquitination is an important chromatin modification with regulatory roles in plant defense against necrotrophic fungi most likely through modulation of gene expression.

Figure 1.

HUB1 Contributes to B. cinerea Resistance. (A) and (B) Disease symptoms (A) and RNA gel blot (B) showing fungal growth after inoculation with 2.5 × 10⁵ Botrytis spores/mL. (C) to (E) Disease symptoms (C), fungal growth (D), and percentage of decayed plants (E) after inoculation with 3.5 × 10⁵ Botrytis spores/mL. The disease assays were done by spray inoculation of Botrytis spores. In (B), total RNA (10 μg) was loaded per lane. In (D), the data show the qPCR amplification of B. cinerea ActinA relative to the Arabidopsis Actin2 gene. In (E), plants were considered decayed when they were completely rotten due to infection. The data represent the mean ± se from a minimum of 26 plants. The experiments were repeated at least three times with similar results. Analysis of variance and Duncan's Multiple Range Test were performed to determine the statistical significance of the differences between the mean values using SAS software (SAS Institute, 1999). The mean values followed by different letters are significantly different from each other (P = 0.05). These experiments were repeated at least three times with similar results. Bc ActinA, Botrytis cinerea ActinA gene; At Actin, Arabidopsis Actin2 gene; d, days after inoculation.

Figure 2.

HUB1 Is Required for Resistance to A. brassicicola. Disease assays on hub1 and 35S:HUB1 plants showing disease symptoms (A), disease lesion size ([B]; top panel) and fungal growth (bottom panel) 6 DAI with A. brassicicola. The disease assays were done by drop inoculation of 5 × 10⁵ A. brassicicola spore/mL. Data points represent average ± se from a minimum of 30 disease lesions. A. brassicicola growth was determined using qPCR amplification of the fungal Cutinase DNA (Ab CutA). The relative DNA levels were calculated by the comparative cycle threshold method (Applied Biosystems) with Arabidopsis Actin2 as the endogenous reference for normalization as described (Bluhm and Woloshuk, 2005). The statistical significance of the differences in the mean values was analyzed as described in the legend for Figure 1. These experiments were repeated at least three times with similar results. Ab CutA, A. brassicicola cutinase gene; At Actin, Arabidopsis Actin2 gene.

Figure 3.

HUB1 Regulates Flowering Time in Arabidopsis. (A) Number of rosette leaves on plants grown under different photoperiods indicative of early flowering in hub1. (B) Expression of flowering genes. (C) and (D) Susceptibility of hub1 plants grown under short days to A. brassicicola (C) and B. cinerea (D). In (A), analysis of variance was performed to determine the statistical significance of the differences between mean numbers of rosette leaves using SAS software (SAS Institute, 1999). Means with different letters are significantly different from each other (P = 0.05). In (B), quantitative RT-PCR was used to determine the expression of flowering genes relative to Actin2 gene. Experiments were repeated at least three times with similar results. d, days after inoculation.

Figure 4.

hub1 Plants Show Reduced Cell Wall Thickness in Epidermal Tissues but Increased Callose Accumulation at the Site of Fungal Infections. (A) Representative pictures showing the size of epidermal cell wall. (B) Mean thickness of epidermal cell walls. (C) and (D) hub1 plants show increased callose accumulation at the site of A. brassiciola (C) and B. cinerea (D) inoculation. In (A), the bars = 200 nm. In (B), the data represent mean ± se from 20 samples. The experiment was repeated twice. The statistical significance of the differences in the mean thickness of the cell wall was analyzed as described in the legend for Figure 1. Means followed by different letters are significantly different from each other (P = 0.05). In (C) and (D), the callose staining assays were from plants 2 d after drop inoculation with 5 × 10⁵ A. brassicicola spores/mL (middle) or 5 × 10⁴ Botrytis spores/mL. The callose data shown in (C) and (D) were quantified using an image analysis program as described in Methods.

Figure 5.

Global and Locus-Specific Histone H3 Methylation Is Not Altered in hub1 Plants. (A) Immunoblot showing global H3K4 methylation. Histone-enriched protein was extracted and immunoblotted using antibodies specific to methylated histones H3K4 methylation. Histone H3 total protein was used as a loading control. (B) ChIP qPCR analysis of histone H3K4 trimethylation and H3K9 dimethylation at the MAF1 and MAF4 promoters. ChIP results for histone H3K4 trimethylation and H3K9 dimethylation at the MAF1 and MAF4 promoters are represented relative to input; the error bars indicate the se. The variation between the samples is very likely due to the overall low level of histone modification as only slightly higher levels than background could be detected. (C) ChIP analysis of control sequences. A heterochromatin control (At4g03770.2, a Gypsy-like retrotransposon) reacts with antibody to H3K9me2, whereas a control for euchromatin (At4g04040, a putative phosphofructokinase beta subunit) reacts with antibody to H3K4me3. Mock, no-antibody control.

Figure 6.

HUB1 Functions Independently of JA-Mediated Defense, but SA Modulates Pathogen Growth in the hub1 Mutant. (A) and (B) A. brassicicola disease assays showing disease symptoms (A), size of disease lesion ([B]; left), and pathogen growth (right) on hub1, sid2, pad4, and double mutants. (C) and (D) Disease symptoms (C), disease lesion size ([D]; left), and pathogen growth on hub1, coi1, and hub1 coi1 plants. All disease assays were repeated at least twice. The disease response data in (A) and (B) are from 5 DAI, whereas those in (C) and (D) are from 2 DAI. The values in (B) and (D) represent mean ± se from at least 30 lesions. h1, hub1-6; h1s2, hub1 sid2; h1p4, hub1 pad4; h1c1, hub1 coi1. Fungal growth was assessed using qPCR amplification of the A. brassicicola cutinase DNA as described in the legend for Figure 1 and in Methods.

Figure 7.

Ethylene Signaling Promotes hub1 Susceptibility to A. brassicicola but Is Required for Resistance to Botrytis. Disease assays on hub1, ein2, and hub1ein2 double mutant showing A. brassicicola disease symptoms (A), size of disease lesion ([B]; left) and fungal growth (right), B. cinerea disease symptoms (C), and fungal growth (D). In (D), total RNA (10 μg) was loaded per lane. All disease assays were repeated at least twice. Data in (B) represent mean ± se from at least 30 lesions. Bc ActinA, B. cinerea ActinA gene; d, days after inoculation.

Figure 8.

HUB1 Is Induced at the Site of Infection by Fungal Pathogens. Expression of HUB1-GUS promoter fusion in responses to mock (A), Botrytis (B), A. brassicicola (C), E. cichoracearum (D), and C. carbonum (E) (Tox+) inoculation. d, days after inoculation.

Figure 9.

The RING E3 Ligase HUB1 Interacts with the MED21 Subunit of Arabidopsis Mediator Complex. (A) HUB1 interacts with MED21 in the yeast two-hybrid assay. Yeast strains containing the MED21 in the prey vector (pAD-MED21) and HUB1 in the bait prey (pBD-HUB1) were assayed for growth on selective medium (-Leu, -Trp, and -His) (left) and β-galactosidase activity (right) showing interaction between MED21 and HUB1 in yeast. The β-galactosidase activity was assayed from yeast cells grown on synthetic complete medium. The positive (+) and negative (−) controls from the Stratagene kit were also assayed in parallel. (B) HUB1 interacts with MED21 in vivo. Bimolecular fluorescence complementation assay showing in vivo interaction between HUB1 and MED21. pHUB1-cYFP and pMED21-nYFP were transiently coexpressed or were coexpressed with the vector alone in N. benthamiana leaf cells. YFP fluorescence was detected when pHUB1-cYFP was coexpressed with pMED21-nYFP. Images were examined under the bright field (left column), fluorescence (YFP), and as a merged image (bottom) showing either no interaction or interaction in the nucleus. DAPI, 4',6-diamidino-2-phenylindole. (C) Siliques from wild-type, MED21/med21, and med21/med21;35S:MED1 lines. Arrows indicate aborted embryos.

Figure 10.

Arabidopsis MED21 Is Required for Resistance to A. brassicicola. (A) and (B) Quantitative RT-PCR showing the expression of MED21 in transgenic MED21 RNAi (A) and 35S:MED21 (B) lines. (C) to (E) Disease symptoms (C), size of disease lesion (D), and fungal growth (E) in MED21 RNAi and overexpression lines after A. brassicicola inoculation. The quantitative RT-PCR data represent the mean ± se from three replicates. In (C), the data are the mean ± se from a minimum of 20 disease lesions. In (E), fungal growth in inoculated plants was determined using the amplification levels of A. brassicicola CutinA (Ab cutA) relative to the Arabidopsis Actin2 gene (At Actin). MO, MED21 overexpression lines; Mi, MED21 RNAi lines. In all cases, the experiments were repeated at least twice.

Figure 11.

Induction of Defense Genes, HUB1 and MED21, by Chitin and B. cinerea. Expression of PR-1 and PDF1.2 genes during B. cinerea infection (A), HUB1 and MED21 upon exposure to chitin (B), Chitinase, osmotin like, and PR3 genes by B. cinerea infection (C), and Arabidopsis defense-related and WRKY transcription factor genes upon exposure to chitin (D). For the RNA blot in (A), total RNA (10 μg) was loaded per lane. In (B) to (D), RT-PCR was performed as described in Methods with 28 cycles, which was within the linear range of amplification. The experiments were repeated at least three times with similar results. Arabidopsis Actin2 and Ubiquitin genes were used as constitutive controls. UBQ, Arabidopsis ubiquitin; d, days after inoculation; h, h after treatment.