New Phytologist Supporting Information

Article title: Arabidopsis downy mildew effector HaRxLL470 suppresses plant immunity by
attenuating the DNA-binding activity of bZIP transcription factor HY5
Authors: Shuyun Chen1*, Tao Ma1*, Shiren Song1, Xinlong Li1, Peining Fu1, Wei Wu1, Jiaqi
Liu1, Yu Gao1, Wenxiu Ye1, Ian B. Dry2 and Jiang Lu1
Article acceptance date: 01 February 2021

The following Supporting Information is available for this article:

Fig. S1 Phylogenetic analysis, sequence alignment and synteny of HaRxLL470 and

RxLL470-Like candidate effectors across different oomycetes.

(a) Phylogenetic tree analysis of HaRxLL470 and RxLL470-like candidate effectors across
oomycete species. Protein sequences were derived and aligned for HaRxLL470/Noco2 and
RxLL470-Like candidate effectors across oomycete species. The alignment was used to build a
phylogenetic tree by using MEGA4 software. The scale represents amino acids substitutions.
(b) Alignment of amino acid sequences of several conserved effector candidates across oomycete
species. SP, signal peptide; RxLR, RxLR motif; TPR_12, Tetratricopeptide repeat 12; NLS,
nuclear localization signal.
(c) Synteny of HaRxLL470 and putative HaRxLL470 orthologs of Phytophthora species. The
synteny analysis was performed by using fungidb. org.
Fig. S2 Alignment of amino acids of HaRxLL470 cloned from Hpa Noco2/Waco9 and

HaRxLL470b translation of the predicted nucleotide sequence in Hpa Emoy Genome

v8.3.2.

Fig. S3 HaRxLL470 expression pattern analysis and phenotype test of HaRxLL470

transgenic lines

(a) Expression pattern of HaRxLL470 during Hpa Noco2 infection. 10-d-old Arabidopsis seedlings
of WT (Col-0) were inoculated with Hpa Noco2 and infected tissues were harvested and analyzed
by qRT-PCR at different time points. Hpa Actin served as an internal control. Data are shown as
the mean of biological triplicates ±SD (n=3).
(b) Western blot analysis of HaRxLL470-YFP expression level with anti-GFP antibody in WT and
HaRxLL470 transgenic lines. Actin was used to confirm equal sample loading.
(c-d) Hypocotyl length test of WT and HaRxLL470 transgenic lines. Arabidopsis Seedlings were
grown with white light for 7 days and hypocotyl length were measured. Data are shown as means
±SD (n=30). Asterisks represent significant difference at p < 0.01 with Student’s t test. Scale bar,
10 mm.
(e) Western blot analysis of estradiol-induced Flag-HaRxLL470 expression level with anti-Flag
antibody in ED-HaRxLL470 transgenic lines. 10-d-old Arabidopsis seedlings were treated with
mock/10 μM β-estradiol for 24 h, and harvested for protein extraction and western blot test. Actin
was used to confirm equal sample loading.
(f) Western blot analysis of Myc-HY5 expression level with anti-Myc antibody in WT and 35S-
HY5 transgenic lines. Actin was used to confirm equal sample loading.
Fig. S4 Effect of HaRxLL470 expression in plant susceptibility to different plant pathogens

(a) HaRxLL470 expression promotes Phytophthora capsici (P. capsici) infection. GFP and GFP-
HaRxLL470 were transiently expressed in N. benthamiana leaves. Leaves were inoculated with P.
capsici after infiltration for 24 h. The lesion areas were photographed at 54 hpi (hours post
inoculation). Scale bar, 10 mm.
(b) Western blot of GFP and GFP- HaRxLL470 expression level with anti-GFP antibody in N.
benthamiana leaves. Actin was used to confirm equal sample loading.
(c) Statistical analysis of the lesion areas in N. benthamiana leaves at 54 hpi. Data are shown as
means ±SD (n=6). Asterisks represent significant difference at p < 0.05 with Student’s t test.
(d) Schematic representation of constructs. The AvrPto promoter were used to drive the expression
of HaRxLL470-His and GFP-His (control). AvrPto1-45 was used to direct the secretion of
HaRxLL470-His/ GFP-His through Type III secretion system (TTSS).
(e) Secretion of the fusion protein HaRxLL470-His and GFP-His by Pseudomonas syringae pv.
Tomato (Pst) DC3000 (HaRxLL470) / Pst DC3000 (GFP). Pst DC3000 (HaRxLL470)/ Pst
DC3000 (GFP) were grown in MM containing tetracycline and fructose (fructose was used as
carbon source). Proteins of the cell-bounds and supernatant were extracted. The expression and
secretion of GFP-His and HaRxLL470-His were analyzed by Western blot with anti-His antibody.
(f) Pst DC3000 (HaRxLL470) population in leaves of wild-type (Col-0) plants. The 4-week-old
plants were inoculated with indicated strains at OD600=0.001. The growth of bacteria was measured
at 3 hours post (0 day) inoculation and 3 days post inoculation (dpi). Data are shown as means ±
SD (n=6). Different letters represent significant differences (p <0.01, Tukey’s LSD test).

Fig. S5 MBP pull-down assay of PiRxLL470-like and NbHY5

In vitro MBP pull-down assay of GST-PiRxLL470-like and MBP-NbHY5. GST-PiRxLL470-like

and MBP-NbHY5 were successfully expressed in E. coli. MBP-NbHY5 served as bait and GST-
PiRxLL470-Like served as prey (GST was used as negative control). GST-PiRxLL470-like pulled
down by MBP-NbHY5 was detected by anti-GST antibody. CBB staining, Coomassie brilliant
blue staining.
Fig. S6 Trypan blue staining of WT, hy5 mutant and 35S-HY5 transgenic lines with

different treatment.

2-week-old Arabidopsis seedlings of indicated genotypes were mock inoculated (H2O) or

inoculated with Hpa Noco2. The plants were harvested and trypan blue staining was performed at
7 dpi. Scale bars, 1 cm.

Fig. S7 EMSA and DNA-protein pull-down assays for identification of HY5 DNA-binding

activity, and Western blot analysis of different proteins transiently expressed in N.

benthamiana

(a) EMSA assay identification of HY5 DNA binding activity to the G-box in the EDS1 promoter.
The EMSA assay was performed with the fused protein MBP-HY5 and biotin-labeled G-box
contained sequence in the EDS1 promoter (Biotin-pEDS1). MBP was used as negative control.
Cold probe, 50×cold competitor DNA relative to the amount of Biotin-pEDS1 probe.
(b) DNA-protein pull-down assay of HY5 DNA binding activity. DNA-protein pull-down assay
was performed with MBP-HY5 and Biotin-pEDS1. MBP was used as negative control.
(c) Structure of the EDS1 promoter-driven dual-luciferase (LUC) reporter gene: (from left to right)
The 35S promoter (35S), REN luciferase (REN), EDS1 promoter (ProEDS1), firefly LUC (LUC),
and nopaline synthase terminator (nos).
(d) Subcellular localization of the recombinant protein Myc-VP16AD-GFP-NLS by transiently
expressed in N. benthamiana (GFP was used as control). The results showed that the recombinant
protein Myc-VP16AD-GFP-NLS was localized in the nucleus. Scale bars, 20 μm.
(e) Western blot of Myc-VP16AD-HY5, Myc-HY5 and Myc-VP16AD-GFP-NLS expression
levels in N. benthamiana leaves using anti-Myc antibody. Actin was used to confirm equal sample
loading.
(f) Western blot of Myc-VP16AD-HY5 and Myc-HY5 expression level with anti-Myc antibody
and GFP and HaRxLL470-GFP expression level with GFP antibody in N. benthamiana leaves.
Actin was used to confirm equal sample loading. The extra bands labelled by anti-GFP antibody
were non-special cross-reacting proteins.
Fig. S8 HaRxLL470 has no detectable influence on the interaction between HY5 and

HDA15.
(a) In vitro pull-down assay of MBP-HY5 and HDA15-His. MBP-HY5 and HDA15-His were
successfully expressed in E. coli. HDA15-His served as bait, MBP-HY5 served as prey and MBP
served as control. MBP-HY5 pulled down by HDA15-His was detected by anti-MBP antibody.
(b) In vitro His pull-down assay suggested that HaRxLL470 has little influence on the interaction
between HY5 and HDA15. 10× and 20× GST-HaRxLL470 indicated the amount of GST-
HaRxLL470 relative to the initial amount of GST-HaRxLL470 protein (1×). 10 × and 20 × GST
indicated the amount of GST relative to the initial amount of GST protein (1×).

Fig. S9 Cluster analysis of differentially expressed genes in hy5 mutant and HaRxLL470

transgenic plants and RNA-seq results validated by qRT-PCR

(a) Cluster analysis of differentially expressed genes (DEGs) in hy5 mutant and HaRxLL470
transgenic plants 3 dpi with Hpa Noco2. Red and green colors in the heatmap represent induced
and repressed genes, respectively. Scale denotes the log2 fold change value.
(b) 12 up-regulated genes were randomly selected and tested by qRT-PCR for validating RNA-
seq results. Data are shown as the mean of biological triplicates ±SD (n=3).
(c) 12 down-regulated genes were randomly selected and tested by qRT-PCR for validating RNA-
seq results. Data are shown as the mean of biological triplicates ±SD (n=3).

Fig. S10 PR1, PR2 and ICS1 qRT-PCR analysis and Yeast One-hybrid assay

(a) PR1, PR2 and ICS1 were down-regulated in hy5 and HaRxLL470 transgenic line after
inoculated with Hpa Noco2. Infected tissues were harvested at 3 dpi and analyzed by qRT-PCR.
Data are shown as means ±SE (n=3).
(b) Yeast one-hybrid assay suggested that HY5 binds to the promoters of PR1, PR2 and ICS1 in
vitro. The blue precipitates on the plates represent the β-galactosidase activities.
Fig. S11 Western blot and phenotype analysis of HaRxLL470 transgenic lines in indicated

background

(a-b) Hypocotyl length analysis of WT, hy5 mutant, hyh mutant, hy5 hyh double mutant and
HaRxLL470 transgenic lines. Arabidopsis Seedlings were grown in white light for 7 days, and
hypocotyl length was measured respectively. Data are shown as means ± SD (n=30). Different
letters represent significant differences (p <0.01, Tukey’s LSD test). Scale bar, 10 mm.
(c) Western blot analysis of HaRxLL470-YFP expression level with anti-GFP antibody in 35S:
HaRxLL470-YFP/hy5. Actin was used to confirm equal sample loading.
(d-e) Hypocotyl length test of indicated plants. Arabidopsis Seedlings were grown on white light
for 7 days and hypocotyl length were measured. Data are shown as means ±SD (n=30). Different
letters represent significant differences (p <0.01, Tukey’s LSD test). Scale bar, 10 mm.
(f) Western blot analysis of HaRxLL470-YFP expression level with anti-GFP antibody in 35S:
HaRxLL470-YFP/hy5 hyh. Actin was used to confirm equal sample loading.
(g-h) Hypocotyl length test of indicated plants. Arabidopsis Seedlings were grown on white light
for 7 days and hypocotyl length were measured. Data are shown as means ±SD (n=30). (p
<0.01, Tukey’s LSD test). Scale bar, 10 mm.
Table S1 List of primers used in this study (see separated Excel file)

Table S2 Amino acid divergence among HaRxLL470 and RxLL470-like effector among

different oomycete species

Protein Identity similarity

PHYSODRAFT_361266 (Phytophthora sojae) 80% 90%

PITG09585 (Phytophthora infestans) 76% 87%

Pv00097 (Plasmopara viticola) 71% 84%

XP_008893349 (Phytophthora parasitica) 76% 88%

XP_024574230 (Plasmopara halstedii) 65% 80%

OWZ00506 (Phytophthora megakarya) 77% 89%

PHYCA_101423T0 (Phytophthora capsici) 77% 90%

POM66359 (Phytophora palmivora) 78% 90%

Table S3 Putative candidates for HaRxLL470 interactors during N. benthamiana IP-mass

analyses (see separated Excel file)

Table S4 Mass-spectrometry identification of NbHY5 peptides

Five peptides of NbHY5 were identified by LC-MS/MS.

pep_seq pep_res_beforeC pep_delta pep_rank pep_exp_mz pep_exp_mr

VKELETK R 0.0179 3 423.7412 845.4679
SPADKENKR R 0.0288 4 522.7896 1043.565
AYLIDLEAR K 0.0295 2 532.3075 1062.6
AYLIDLEAR K 0.0322 2 532.3088 1062.603
GRSPADKENK R 0.0447 5 551.3083 1100.602
SSSSALHHELK
R 0.0411 1 786.3743 2356.101
EGMESDDEIR
Table S5 List of differentially expressed genes (DEGs) in hy5 mutant during Hpa Noco2

inoculated at 3 dpi (see separated Excel file)

Table S6 List of differentially expressed genes (DEGs) in HaRxLL470 transgenic line

during Hpa Noco2 inoculated at 3 dpi (see separated Excel file)

Table S7 List of differentially expressed HY5-targeted candidate genes in hy5 mutant

and/or HaRxLL470 transgenic plants (see separated Excel file)

Methods S1 Secretion and protein assay by Type III secretion system

Effector protein secretion and detection by Pst DC3000 type III secretion system (TTSS) were
performed as previously described (Huynh et al., 1989; van Dijk et al., 1999; Collmer et al., 2000).
The first 15 codons of AvrPto are sufficient to direct the secretion of the foreign effector protein
(Collmer et al., 2000). Hence, the DNA fragment of the promoter and the first 45 bp of the coding
sequence of AvrPto were amplified by genomic DNA extracted from Pst DC3000 and cloned into
a pUCP19 vector. The DNA fragment of GFP and ΔSP-HaRxLL470 were cloned and inserted into
the modified pUCP19 vector which was bearing 6×His tag (ProAvrPto:AvrPto1-45-GFP-6×His

and ProAvrPto:AvrPto1-45-HaRxLL470-6×His). The vector constructs were transformed into Pst

DC3000 competent cell using an electric current. The culture and supernatant pooling of Pst
DC3000 with GFP/HaRxLL470 were performed as previously described (Huynh et al., 1989).
Cultures were centrifuged at 4 °C to separate into cell-bound and supernatant fractions for protein
extraction. Proteins of cell-bound and supernatant fractions were extracted as previously describe
(Ham et al., 1998) and confirmed by western blot using anti-His antibody (TransGen Biotech Co.
Ltd., Beijing, China).

Methods S2 Yeast signal sequence trap system assay

The signal peptide of HaRxLL470 was predicted by the SignalP and TargetP (Emanuelsson et al.,
2007). The yeast signal sequence trap system assays were performed as previously described (Oh
et al., 2009). Briefly, the DNA fragment of the predicted HaRxLL470 signal peptide (SP) and the
next two amino acids was cloned and inserted into pSUC2T7M13ORI (pSUC2) bearing the
truncated invertase gene SUC2, lacking Met and the SP (Jacobs et al., 1997). The construct was
transformed into the invertase-negative yeast strain YTK12 and invertase secretion detection was
performed as previously described (Oh et al., 2009).

Methods S3 Yeast one-hybrid assay

The full-length HY5 coding sequence was cloned from Arabidopsis thaliana ecotype Col-0 and

inserted into pB42AD (Clontech Laboratories) to generate pB42AD-HY5 after digested with

appropriate restriction enzymes. The sequences of cis-acting element listed in Table S7 were

introduced into pLaczi (Clontech Laboratories). The constructs were co-transformed into EGY48

yeast cells using the lithium acetate transformation procedure, which was described in the Yeast

Protocols Handbook (Clontech Laboratories).

Methods S4 Confocal microscopy assay

Constructs containing nYFP-HaRxLL470, HY5-cYFP, HYH-cYFP, and cYFP were transformed

into A. tumefaciens strain GV3101. The cultures contained nYFP- HaRxLL470/nYFP-HaRxL9
were mixed with HY5-cYFP/ HYH-cYFP/ cYFP. Constructs containing HaRxLL470-GFP and
RFP was transformed into A. tumefaciens strain GV3101. The cultures contained HaRxLL470-
GFP was mixed with RFP. The mixture was infiltrated into N. benthamiana leaves using needle-
free syringe. Fluorescence signals were detected with a confocal microscope (Leica TCS SP5II;
Leica Microsystems, Wetzlar, Germany) 48 h after inoculation. YFP was excited by using a 510
nm argon laser and fluorescence was captured by 514 nm filter. mCherry was excited by using a
543 nm argon laser and fluorescence was captured by 600-650 nm filter. Data was processed using
LSM software.

Methods S5 Immunoprecipitation for mass spectrometry (IP-MS)

IP-MS assay was performed as previously described (Derkacheva et al., 2013) with minor
modification. In general, HaRxLL470-Flag/ Luciferase-Flag (used as negative control) was
transiently expressed in N. benthamiana leaves. Ten g leaves were harvested and ground in a
mortar with liquid nitrogen. Soluble proteins were extracted in lysis buffer (50 mM Tris-HCl, 150
mM NaCl, and 0.2% (v/v) Triton-X-100 and protease inhibitors; pH 7.5) and incubated with
prewashed anti-Flag magnetic beads (New England Biolabs, Ipswich, MA, USA) on a rotator at
4 °C for 90 min. The beads were washed 5 times by using lysis buffer (50 mM Tris-HCl, 150 mM
NaCl, and 0.2% (v/v) Triton-X-100; pH 7.5) and boiled with 30 μL 1.5×sodium dodecyl sulphate
(SDS) loading buffer at 100 °C for 5 min. Immunoprecipitates were detected by 12% SDS-PAGE
electrophoresis and Coomassie bright blue (CBB) staining.

The specific bands were excised and in-gel digestion was performed as previously described
(Shevchenko et al., 1996). In brief, the gel was washed twice using ultrapure water and decolored
using destaining solution (50% acetonitrile, 25 mM ammonium bicarbonate). Dehydrated gel was
reduced using 10 mM dithiothretol (DTT) for 1 h at 56 °C and then alkylated in 55 mM
iodoacetamide (IAM) for 45 °C at room temperature in the dark. The gel was washed twice using
25 mM ammonium bicarbonate and then washed twice using destaining solution. The dehydrated
gel was digested using 12.5 ng/μl Trypsin overnight at 37 °C. The digestion was terminated using
0.1% FA. 10 μl sample was detected for LC-MS/MS analysis using a Q-Exactive mass
spectrometer (Thermofisher Scientific). Searches were performed using Mascot software (Matrix
Science, London, UK) against a database containing Solanaceae sequence (Solanaceae_uni_4070
4070). The search parameters were: type of search, MS/MS ion search; enzyme, trypsin; fixed
modification, carbamidomethyl (C); variable modifications, Gln->pyro-Glu (N-term Q), oxidation
(M); Mass values, monoisopic; peptide mass tolerance, 0,05 Da; fragment tolerance, 0.01 Da; max
missed cleavages, 2. This experiment was repeated two times. Proteins were taken into
consideration when at least two unique peptides were identified.

Methods S6 Hypocotyl length measurement

To measure the hypocotyl length, the seedlings were grown on Murashige and Skoog (MS)
medium containing 1% (w/v) sucrose and 0.8% (w/v) agar for 7 d under a long-day cycle (14 h
day/10 h night). The hypocotyls were photographed and measured with NIH ImageJ
(http://rsbweb.nih.gov/ij/).

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