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. 2015 Feb 3;10(2):e0117067.
doi: 10.1371/journal.pone.0117067. eCollection 2015.

Arabidopsis HFR1 is a potential nuclear substrate regulated by the Xanthomonas type III effector XopD(Xcc8004)

Choon Meng Tan  1 , Meng-Ying Li  2 , Pei-Yun Yang  2 , Shu Heng Chang  2 , Yi-Ping Ho  2 , Hong Lin  2 , Wen-Ling Deng  3 , Jun-Yi Yang  4
Affiliations

Arabidopsis HFR1 is a potential nuclear substrate regulated by the Xanthomonas type III effector XopD(Xcc8004)

Choon Meng Tan et al. PLoS One. .

Abstract

XopDXcc8004, a type III effector of Xanthomonas campestris pv. campestris (Xcc) 8004, is considered a shorter version of the XopD, which lacks the N-terminal domain. To understand the functions of XopDXcc8004, in planta, a transgenic approach combined with inducible promoter to analyze the effects of XopDXcc8004 in Arabidopsis was done. Here, the expression of XopDXcc8004, in Arabidopsis elicited the accumulation of host defense-response genes. These molecular changes were dependent on salicylic acid and correlated with lesion-mimic phenotypes observed in XVE::XopDXcc8004 transgenic plants. Moreover, XopDXcc8004 was able to desumoylate HFR1, a basic helix-loop-helix transcription factor involved in photomorphogenesis, through SUMO protease activity. Interestingly, the hfr1-201 mutant increased the expression of host defense-response genes and displayed a resistance phenotype to Xcc8004. These data suggest that HFR1 is involved in plant innate immunity and is potentially regulated by XopDXcc8004.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Salicylic acid-dependent defense responses were elicited by the expression of XopDXcc8004 in Arabidopsis.
(a) Schematic representation of XopD proteins. (b) Morphological examination and trypan blue staining of two-week-old leaves of Arabidopsis XVE::XopD Xcc8004 transgenic plants. Lesion-mimic phenotypes were indicated by arrows. Scale bar: 1 mm. (c) Translated products of XopD Xcc8004 and XopD Xcc8004 (C355A) were examined by western blotting using a specific antibody against XopDXcc8004. Anti-tubulin was used for loading control. (d) The expression levels of genes involved in the SA-mediated defense signaling network were examined by qRT-PCR and normalized to EF1α. The relative expression levels of each gene in the DMSO control were set at 1.
Figure 2
Figure 2. XopDXcc8004 suppresses the virulence of the Xcc8004.
(a) Bacterial growth of the Xcc8004 strain in Arabidopsis transgenic plants. DMSO or β-estradiol was applied 24 h before bacterial inoculation. Hand-infiltrated leaves were collected at the indicated times for measuring the in planta growth of bacterial populations. (b) Bacterial growth of Xcc8004 spp. in Arabidopsis plants were measured to examine the effects of XopDXcc8004 on the virulence of Xcc8004. Statistically significant differences were determined using one-way ANOVA (* indicates p < 0.05).
Figure 3
Figure 3. XopDXcc8004(C355A) loses the activity for activating plant immunity.
(a) Morphological examination and trypan blue staining of two-week-old leaves of Arabidopsis XVE::XopD Xcc8004 (C355A) transgenic plants. Scale bar: 1 mm. (b) The expression levels of genes involved in the SA-mediated defense signaling network were examined by qRT-PCR and normalized to EF1α. The relative expression levels of each gene in the DMSO control were set at 1.
Figure 4
Figure 4. Expression of XopDXcc8004 induces a long hypocotyl phenotype in Arabidopsis.
(a) Phenotypes of 12-day-old Arabidopsis transgenic plants carrying a XopD Xcc8004 gene driven by the inducible XVE promoter. Scale bars: 8 mm. (b) Average hypocotyl lengths of seedlings grown on medium containing DMSO or 20 μM β-estradiol. Statistically significant differences were determined using one-way ANOVA (** indicates p < 0.005).
Figure 5
Figure 5. XopDXcc8004 interacts with HFR1.
(a) Investigation of the interaction between HFR1 and XopDXcc8004 by yeast two-hybrid analysis. Yeast strains transformed with two plasmids (prey and bait) were plated onto synthetic-defined (SD) minimal medium lacking tryptophan/leucine or tryptophan/leucine/histidine. (b) Nicotiana benthamiana leaves were co-infiltrated with agrobacterium carrying 35S::XopD Xcc8004 -YFP and 35S::HFR1-CFP. Fluorescence and differential interference contrast (DIC) images were obtained by confocal laser scanning microscopy. 35S::YFP and 35S::CFP were used for control. Scale bars: 15 μm. (c) Investigation of the interaction between HFR1 and XopDXcc8004 by using pull-down assays. Briefly, 2 μg of GST or GST fusion proteins were used to pull down 2 μg of MBP or MBP fusion proteins, and an anti-MBP antibody was used to detect the associated proteins by western blotting (the left panel). The right panels present the input proteins examined by anti-MBP or anti-GST antibodies. The asterisk indicates the protein signal of MBP-HFR1.
Figure 6
Figure 6. K72 in HFR1 is desumoylated by XopDXcc8004 in vitro.
(a) The deduced amino acid sequence of HFR1 from 65 to 80 a.a. The arrow indicates a putative sumoylation site at K72 of the consensus motif ΨKxE/D of HFR1. (b) An in vitro sumoylation system was established by using purified recombinant proteins, including Arabidopsis SAE1, SAE2, SCE1, and AtSUMO1. MBP-HFR1 or MBP-HFR1(K72A) were used as potential substrates for sumoylation and detected with an anti-MBP antibody. (c) Together with SAE1, SAE2, SCE1, AtSUMO1, and MBP-HFR1, XopDXcc8004 was added to the reaction mixture to investigate the sumoylation of HFR1. XopDXcc8004(C355A), a SUMO protease mutant, was used as control. Asterisks indicate sumoylated MBP-HFR1 proteins. Arrowheads indicate unmodified MBP-HFR1 proteins. Signals below the unmodified MBP-HFR1 proteins were degraded products of purified MBP-HFR1 proteins.
Figure 7
Figure 7. hfr1–201 increases plant immunity against Xcc8004 spp.
(a) Arabidopsis WT and hfr1–201 mutant plants treated with (8 hr) or without (0 hr) 2 mM SA were collected for total RNA extraction. The expression levels of genes involved in the SA-mediated defense signaling network were examined by qRT-PCR and normalized to EF1α. The relative expression levels of each gene in the WT plants without SA treatment were set at 1. (b) Bacterial growth in Arabidopsis WT and hfr1–201 mutant plants were measured to examine the effects of HFR1 on the resistance of Arabidopsis against Xcc8004 spp. Hand-infiltrated leaves were collected at the indicated times for measuring the in planta growth of bacterial populations. Statistically significant differences were determined using one-way ANOVA (* indicates p < 0.05).
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