Pha13 is involved in expression of SA-induced PhaNPR1

Pha13 (Orchidstra 2.0 database, http://orchidstra2.abrc.sinica.edu.tw, accession number PATC148746, the Pha13 coding sequence can be found in S1 File) was identified here for the first time using the CymMV-VIGS system in Phalaenopsis aphrodite. Our data indicated that silencing Pha13 decreased the RNA of PhaPR1 (Fig 1A). Amino acid sequence analysis revealed that Pha13 contained A20 and AN1 domains (Fig 1B–1D) and belonged to a fast emerging class of zinc-finger proteins (ZFPs), termed stress associated proteins (SAPs) in plants [28].

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Fig 1. Identification of Pha13 involvement in PhaPR1 expression by CymMV-based VIGS system and sequence analysis.

(A) Expression level of Pha13, PhaPR1, and CymMV were analyzed by RT-PCR from leaves of P. aphrodite inoculated with buffer (Mock), or infiltrated with agrobacterium carrying pCymMV (Cy) or pCymMV-Pha13 (Pha13-VIGS). PhaUbiquitin 10 (PhaUBQ10) was used as a loading control, and the relative expression level of corresponding genes are indicated. (B) Domains of Pha13. Open rectangle indicates the entire protein. A20 (black rectangle) and AN1 (diagonally-striped rectangle), nuclear localization signal (open square with vertical lines), and 21-nucleotide position (short black line) used for designing hairpin RNA of Pha13 (hpPha13) are indicated. (C) Sequence alignment of A20/AN1 zinc finger domains of Pha13 with stress-associated proteins from Arabidopsis thaliana (AtSAP5, accession number: AT3G12630), and Oryza sativa (OsSAP3, accession number: LOC_Os01g56040.1; OsSAP5, accession number: LOC_Os02g32840.1) are shown. Conserved amino acid sequences are indicated with a black box. Conserved cysteine (C) and histidine (H) are indicated with a black triangle. (D) Primary sequence organization of A20 and AN1 domains. Xn: the number of amino acid residues between zinc ligands. (B to D) The mutation positions for domain functional analysis are indicated with red triangle.

https://doi.org/10.1371/journal.ppat.1007288.g001

To further address the interrelationship between Pha13 and known SA-related genes, we knocked down Pha13 RNAs by infiltration of agrobacterium carrying the 35S promoter driven hairpin RNA-expressing constructs of phpPha13 in P. aphrodite (Fig 2A). Total RNAs extracted from agrobacterium-infiltrated plants were used to detect RNA expression of Pha13, PhaNPR1, and PhaPR1 by qRT-PCR. Knockdown of Pha13 RNA is correlated with decreased RNA levels of PhaNPR1 and PhaPR1 (Fig 2A). Furthermore, we generated transient overexpression of Pha13 in P. aphrodite by infiltration of agrobacterium carrying the overexpression constructs (pPha13-oe). Transient overexpression of Pha13 in P. aphrodite increased the RNA levels of PhaNPR1 and PhaPR1 (Fig 2B). On the other hand, transient silencing of PhaNPR1 (infiltration of agrobacterium carrying phpPhaNPR1) did not affect the RNA accumulation of Pha13 (Fig 2C). This indicates that Pha13 affects the expression of PhaNPR1 but not vice versa.

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Fig 2. Pha13 is involved in the expression of PhaPR1 and PhaNPR1, and induced by phytohormone treatment.

(A and B) Expression level of Pha13, PhaNPR1, and PhaPR1 were analyzed by qRT-PCR from leaves of P. aphrodite infiltrated with agrobacterium carrying vector (Vector); hairpin RNA (hpRNA) vector to knock down Pha13 (hpPha13; A); and overexpression vector of Pha13 (Pha13-oe; B). The RNA level of vector was set to 1. (C) Transient silencing of PhaNPR1. Expression level of PhaNPR1 and Pha13 were analyzed by qRT-PCR from leaves of P. aphrodite infiltrated with agrobacterium carrying the vector (Vector) or hairpin RNA (hpRNA) vector to knock down PhaNPR1 (hpPhaNPR1). The RNA level of the vector was set to 1. (A to C) Mean ± SD; n = 3 biological replicates; *, P < 0.05, Student’s t-test compared to vector. (D to F) Time-course expression of Pha13 under different plant hormone treatments in P. aphrodite. Expression level of Pha13 was analyzed by qRT-PCR from leaves treated with SA (D), JA (E), and ET (F) at different hours (h) post-treatment. Inoculation buffer treatment was used as a mock control. Results of qRT-PCR were relative to that of mock at individual time course for relative quantification. The RNA level at 0 hour was set to 1 for comparison between different time courses. PhaPR1 and PhaNPR1 were used as SA marker genes. PhaJAZ1 and PhaACO2 were used as JA and ET marker genes, respectively. Data represent mean ± SD; n = 3 technical replicates; *, P < 0.05, Student’s t-test compared to 0 h. One representative experiment is shown from at least three replicates of similar results. PhaUbiquitin 10 was used as an internal control for normalization.

https://doi.org/10.1371/journal.ppat.1007288.g002

The results from our transient silencing and overexpression of Pha13 showed that Pha13 is involved in the SA-related plant immune pathway. Next, we tested whether SA or other plant hormones affect Pha13 transcription. Plant samples were collected at different time points up to 72 h after SA, jasmonic acid (JA), and ethylene (ET) treatment, and analyzed for RNA of Pha13 and corresponding hormone-marker genes by qRT-PCR. Pha13 was induced by SA, JA, and ET at 1 h, 6 h, and 6 h post-treatment, respectively (Fig 2D–2F). The results indicated that post-treatment expression of Pha13 was induced earlier in SA treatment, at 1 h post-treatment, compared to up regulation of PhaNPR1 at 24 h post-treatment (Fig 2D).

Pha13 is induced by CymMV infection and involved in virus resistance

To determine whether Pha13 is involved in plant resistance to virus infection, we first analyzed Pha13 expression in mock- or CymMV-inoculated P. aphrodite by qRT-PCR. The results showed that Pha13 is induced by CymMV (Fig 3A). Furthermore, we assayed the virus accumulation in transient silenced or overexpressed Pha13 in P. aphrodite. Transient knockdown of Pha13 increased CymMV accumulation in infected P. aphrodite (Fig 3B); overexpression of Pha13 in CymMV-infected P. aphrodite decreased CymMV accumulation (Fig 3C). To validate the results, we generated transgenic P. equestris overexpressing Pha13 (35S::FLAG-Pha13). Western blot using antiserum against Pha13 indicated that the overexpressed Pha13 could be detected in every individual asexually propagated progeny derived from transgenic T0 lines 27, 29 and 30 (Fig 3D). When we inoculated CymMV into 3 individual progenies derived from 3 transgenic lines, the results showed that CymMV decreased to a very low level in all progenies of the three transgenic lines as compared to the non-transgenic lines (Fig 3E).

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Fig 3. Pha13 is induced by CymMV and involved in virus accumulation.

(A) The CymMV accumulation level and expression level of Pha13 were analyzed by qRT-PCR in healthy (Mock) and CymMV-infected (Cy-infected) P. aphrodite. The RNA level in the CymMV was set to 1. Data represent mean ± SD; n = 3 biological replicates; *, P < 0.05, Student’s t-test compared to mock. (B and C) Transient silencing or overexpression of Pha13 in CymMV-infected plants. RNA level of Pha13 and CymMV were analyzed by qRT-PCR from leaves of CymMV-infected P. aphrodite infiltrated with agrobacterium carrying vector (Vector); hairpin RNA (hpRNA) vector to knockdown Pha13 (hpPha13; B); or overexpression vector of Pha13 (Pha13-oe; C). The RNA level of vector was set to 1. Data represent mean ± SD; n = 3 biological replicates; *, P < 0.05, Student’s t-test compared to vector. D, The protein level of Pha13 in WT and transgenic P. equestris (Pha13#27, #29 and #30) expressing Pha13, Pha13-oe (35S::Flag-Pha13), were analyzed by anti-Pha13 antibody. The anti-Tubulin antibody (Anti-Tub) was used as a loading control. (E) Three wild-type plants and three plants derived from the same transformed protocorm were used for analysis. RNA level of Pha13 and CymMV were analyzed by qRT-PCR from leaves of WT or transgenic P. equestris (Pha13#27, #29 and #30) inoculated with CymMV. The RNA level of WT was set to 1. Data represent mean ± SD; n = 3 biological replicates; *, P < 0.05, Student’s t-test compared to WT. PhaUbiquitin 10 was used as an internal control for normalization.

https://doi.org/10.1371/journal.ppat.1007288.g003

Pha13 participates in the expression of SA-induced PhaNPR1-dependent and -independent virus resistance

To explore the gene(s) affected by Pha13, we conducted microarray analysis of P. aphrodite overexpressing Pha13 (pPha13-oe). Global gene expression analysis comparing microarray data of plants overexpressing Pha13 to the vector control 5 days post agroinfiltration revealed that overexpression of Pha13 affected the expression of 10639 genes (S1A Fig). Gene ontology of the affected genes indicated that Pha13 is involved in cellular processes, metabolic processes, and single-organism processes (S1B Fig). To verify the microarray data, we analyzed the RNA expression level by qRT-PCR of three genes PhaNPR1, PhaRdR1, and Glutaredoxin (PhaGRX), that were up-regulated in the Pha13 overexpression plants (S2 Fig).

In our analysis of differentially expressed genes of plants overexpressing Pha13, we observed several genes previously reported to be involved in plant resistance. Among them are SA-induced genes that are known to be NPR1-dependent and -independent (S1 Table). We selected two genes, PhaRdR1 and PhaGRX, reported to be involved in plant resistance [29, 30] and induced by SA, for further analysis. One of the selected genes, RdR1 is positively regulated by NPR1 [24], whereas the regulation of GRX is irrelevant to NPR1 in Arabidopsis [31].

In orchids, PhaRdR1 and PhaGRX were both induced by SA and CymMV infection (Fig 4A and 4B). We next investigated whether PhaNPR1 regulates PhaRdR1 and PhaGRX expression in P. aphrodite through transiently delivering PhaNPR1 hairpin RNA into P. aphrodite by agroinfiltration carrying phpPhaNPR1. Our data for orchids is consistent with studies in other systems, in which transient silencing of PhaNPR1 can decrease the downstream marker genes, PhaPR1 and PhaRdR1, whereas PhaGRX remain unchanged (Fig 4C).

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Fig 4. Pha13 is involved in the expression of PhaNPR1-dependent and -independent marker genes.

(A) Expression level of PhaRdR1 and PhaGRX were analyzed by qRT-PCR from leaves of P. aphrodite treated with buffer (Mock) or SA, at different times (h) post-treatment. Results of qRT-PCR were relative to that of mock at individual time course for relative quantification. The RNA level at 0 h was set to 1. Data represent mean ± SD; n = 3 technical replicates; *, P < 0.05, Student’s t-test compared to 0 h. One representative experiment is shown from at least three replicates of similar results. PhaUbiquitin 10 was used as an internal control for normalization. (B) Expression level of PhaRdR1, PhaGRX, and CymMV accumulation level were analyzed by semi-quantitative RT-PCR from leaves of P. aphrodite inoculated with buffer (Mock) or infected with CymMV (Cy). PhaUbiquitin 10 was used as a loading control, and relative expression levels of the corresponding genes are indicated. (C) Expression level of PhaNPR1, PhaPR1, PhaRdR1, and PhaGRX were analyzed by qRT-PCR from leaves of P. aphrodite infiltrated with agrobacterium carrying the vector (Vector) or hairpin RNA (hpRNA) vector to knock down PhaNPR1 (hpPhaNPR1). (D and E) Expression level of PhaRdR1, PhaGRX, and CymMV accumulation level were analyzed by qRT-PCR from leaves of CymMV-infected P. aphrodite infiltrated with agrobacterium carrying vector (Vector); hairpin RNA (hpRNA) vector to knockdown PhaRdR1 (hpPhaRdR1, D), or PhaGRX (hpPhaGRX, E). (F and G) Expression level of Pha13, PhaRdR1, and PhaGRX were analyzed by qRT-PCR from leaves of P. aphrodite infiltrated with agrobacterium carrying vector (Vector) or hairpin RNA (hpRNA) vector to knockdown Pha13 (hpPha13; F), or to overexpress Pha13 (Pha13-oe; G). C to G, The RNA level of vector was set to 1. Data represent mean ± SD; n = 3 biological replicates; *, P < 0.05, Student’s t-test compared to vector. PhaUbiquitin 10 was used as an internal control for normalization.

https://doi.org/10.1371/journal.ppat.1007288.g004

To reveal whether PhaRdR1 or PhaGRX is involved in virus resistance, we transiently silenced the two genes in CymMV pre-infected P. aphrodite. Consistent with previous reports [29], our data indicated that silencing PhaRdR1 (phpPhaRdR1) increases CymMV accumulation (Fig 4D). Although a previous report indicated that GRX is involved in plant defense against Botrytis cinerea infection [30], its role in virus infection remains elusive. Our data indicated that silencing PhaGRX (phpPhaGRX) increased CymMV accumulation (Fig 4E).

Our data suggested that both PhaRdR1 and PhaGRX are involved in SA-induced virus resistance. To further analyze the relationship between Pha13 in the regulation of PhaRdR1 and PhaGRX, we performed transient silencing and overexpression assay of Pha13 in P. aphrodite. Transient silencing Pha13 did not affect the expression of PhaRdR1 or PhaGRX, while transiently overexpressing Pha13 increased the expression of PhaRdR1 and PhaGRX (Fig 4F and 4G). Our data suggested that Pha13 positively regulates PhaRdR1 and PhaGRX expression.

Pha13 AN1 domain is involved in the expression of PhaNPR1, and A20 and AN1 domains are both required for induced expression of PhaRdR1, PhaGRX, and CymMV accumulation

To analyze the function of A20 and AN1 domain in Pha13, we overexpressed wild-type or mutant of Pha13 (mutated A20, and/or AN1 domain of Pha13) in healthy or CymMV pre-infected P. aphrodite. We substituted the conserved cysteine and histidine to glycine on A20 and/or AN1 of Pha13 (Fig 1C and 1D) to generate the A20 domain mutant (Pha13A20m), AN1 domain mutant (Pha13AN1m), and the A20 and AN1 domain double mutant (Pha13A20mAN1m). The results indicated that overexpression of Pha13 A20 mutant (Pha13A20m) increased expression of PhaNPR1, which is consistent with overexpression of Pha13 wild-type (Fig 5A). The stable expression of Pha13 wild-type and mutants were confirmed by immunoblotting analysis (S3 Fig). Overexpression of Pha13 AN1 mutant (Pha13AN1m) in P. aphrodite decreased the expression of PhaRdR1, whereas the expression of PhaNPR1 and PhaGRX remained unchanged (Fig 5A). Overexpression of Pha13 A20 and AN1 mutant (Pha13A20mAN1m) in P. aphrodite decreased the RNA levels of PhaRdR1 and PhaGRX, while the expression of PhaNPR1 remains unchanged (Fig 5A). Overexpression of any Pha13 A20 and/or AN1 mutant resulted in increased accumulation of CymMV (Fig 5B). These data are summarized in Fig 5C and S4 Fig. Our data suggests that Pha13 AN1 domain alone can affect the expression of PhaNPR1, and both A20 and AN1 domains are required for regulation of PhaRdR1, PhaGRX, and CymMV accumulation.

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Fig 5. Pha13 A20 and AN1 domains are involved in the expression of PhaNPR1-dependent and -independent immune gene(s) and virus accumulation.

(A and B) Expression level of Pha13, PhaNPR1, PhaRdR1, PhaGRX, and CymMV accumulation level were analyzed by qRT-PCR of healthy P. aphrodite leaves (A), or CymMV pre-infected P. aphrodite (B) and infiltrated with agrobacterium carrying vector (Vector), overexpression clones of Pha13, or the respective A20 and/or AN1 mutant clones. The RNA level of vector was set to 1. Data represent mean ± SD; n = 3 biological replicates; *, P < 0.05, Student’s t-test compared to vector. PhaUbiquitin 10 was used as an internal control for normalization. (C) A model illustrating SA-induced Pha13 transcriptional and post-translational regulation leading to the activation of immune responsive genes. Virus infection caused accumulation of SA and leads to post-translational modification of NPR1, allowing it to enter into the nucleus for the activation of NPR1-dependent immune responsive genes including PR1 and RdR1. On the other hand, increased SA can also regulate Pha13 at both transcriptional and post-translational level and leads to the expression of NPR1-dependent and independent immune responsive genes including NPR1, RdR1 and GRX for virus resistance.

https://doi.org/10.1371/journal.ppat.1007288.g005

Pha13 contains self-ubiquitination E3 ligase activity and the A20 domain is more important in conferring E3 ligase activity

A20-type zinc finger proteins have been reported to confer ubiquitin ligase activity [32]. Therefore, we analyzed whether Pha13 has self-ubiquitination E3 ligase activity. We purified His-tagged recombinant Pha13 (Pha13-His) from E. coli for self-ubiquitination E3 ligase activity analysis. Self-ubiquitination E3 ligase activity was observed in the presence of human E1 and E2 with Pha13-His (Fig 6) by using anti-FLAG antibody to detect FLAG-ubiquitin. Furthermore, we also analyzed the self-ubiquitination E3 ligase activity of the A20 and/or AN1 domain. As shown in Fig 6, E3 ligase activity of Pha13 was greatly reduced in A20 mutant (Pha13A20m) or A20/AN1 double mutant (Pha13A20mAN1m). AN1 mutant (Pha13AN1m) showed higher self-ubiquitination E3 ligase activity than other domain mutants, but lower than the wild-type form of Pha13 (Fig 6). The results indicate that A20 domain of Pha13 plays a more important role in conferring E3 ligase activity. The data are summarized in S4 Fig.

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Fig 6. Pha13 confers self-ubiquitination E3 ligase and ubiquitin binding ability.

In vitro ubiquitination analysis was performed on recombinant proteins of Pha13, A20 mutant (Pha13A20m), AN1 mutant (Pha13AN1m), A20/AN1 double mutant (Pha13A20mAN1m) with or without FLAG-tagged ubiquitin (FLAG-Ub), ubiquitin-activating enzyme (E1), or ubiquitin-conjugating enzyme (E2). Ubiquitinated proteins were analyzed with immunoblotting using anti-FLAG antibodies. Coomassie staining was used as a loading control. The ubiquitinated Pha13 (Pha13-2Ub), E1 conjugated with one Ub (E1-Ub), E2 conjugated with one or two Ub (E2-Ub, E2-2Ub), and free Ub are indicated with arrow.

https://doi.org/10.1371/journal.ppat.1007288.g006

Pha13 exhibited ubiquitin binding activity

In addition, to search for Pha13 substrate or interacting proteins, we constructed a yeast two-hybrid (Y2H) library with RNA extracted from SA-treated P. aphrodite. In our initial screening, we identified 56 positive clones. After sequencing the clones, we found that the positive clones include 25 clones encoding ubiquitin, 4 clones encoding partial sequences of thioredoxin-like proteins, 3 clones encoding partial DNAJ-like proteins, and the rest of clones encode proteins of different identity that only appear once (S2 Table). The full length of clones encoding proteins appearing more than once in our initial screen were subjected to further Y2H analysis. Only clones encoding ubiquitin showed positive interaction (Fig 7A). To map the ubiquitin binding domain of Pha13, a series of pha13 deletion mutants were generated and used for Y2H assay (Fig 7A). The results indicated A20 domain is mainly involved in ubiquitin binding (Fig 7A).

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Fig 7. Mapping the ubiquitin-binding region in Pha13 by a yeast two-hybrid assay.

(A) Left panel indicating Pha13 putative functional domains (A20 and AN1) and the sites of truncation. The ubiquitin-binding region is mapped to the A20 domain of Pha13. Various combinations of Pha13 truncations cloned in pGBK vector were co-transformed with pGADPhaUb into yeast AH109 strain. The transformants were spotted on control plates (2DO: Leucine-Trptophan dropped out medium) and selective plates (3DO/X-Gal: Leucine-Trptophan-Histidine dropped out and X-Gal added medium), which were incubated at 30°C for 4 days before photography. Red stars indicate cysteine in A20 domain. Yellow stars indicate cysteine replaced with lysine. (B) In vitro pull-down assay on AtSAP5 (used as positive control) [44], Pha13 and Pha13 A20 mutant (Pha13A20m), with polyubiquitin chains.

https://doi.org/10.1371/journal.ppat.1007288.g007

To assay which type of ubiquitin chain binds to Pha13, in vitro pull-down assays were performed by mixing recombinant Pha13, A20 mutant (Pha13A20m), or AtSAP5 (positive control) with commercially available linear polyubiquitin chains. After precipitation, the bounded linear polyubiquitin was detected by immunoblot with the anti-ubiquitin antibody. As shown in Fig 7B, Pha13 has the ability to bind to M1, K6, K11, K29, K33, K48 and K63-linked ubiquitin chains, and A20 domain is mainly responsible for the binding.

SA regulates Pha13 at post-translational level

Our data indicates that Pha13 confers self-E3 ligase activity and ubiquitin chain binding activity. To analyze whether SA stimulate the ubiquitination of Pha13 or binding to ubiquitinated protein, immunoprecipitation (IP) assay was performed to precipitate the Pha13 and ubiquitinated Pha13 in H₂O (Mock) and SA treated P. aphrodite using anti-Pha13 antibody. Then, we used anti-Pha13 or anti-ubiquitin antibody for time course determination of Pha13 and ubiquitinated Pha13 (Fig 8A). We have repeated this experiments for more than 3 times with consistent results. Our data indicates that mock and SA treatment increased accumulation of the Pha13. Compared to mock-treated P. aphrodite, decreased accumulation of Pha13 was observed in SA-treated P. aphrodite at each time point (Fig 8A). When we used MG132 to inhibit 26S proteasome activity, the protein levels of Pha13 increased in SA treated- but not in mock treated-P. aphrodite (Fig 8B).

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Fig 8. SA regulates Pha13 at post-translational level.

(A) Total proteins were extracted from leaves of P. aphrodite without treatment (0 h), treated with H₂O (Mock), or SA at different times (h), and used for in vivo immunoprecipitation (IP) assay using anti-Pha13 antibody. Samples after IP were analyzed by immunoblotting using anti-Pha13 or anti-ubiquitin antibody. (B) P. aphrodite was treated with H₂O (Mock) or SA, and immediately followed by infiltration of DMSO (-) or MG132 (+). Total proteins were extracted from leaves of the treated samples at 3 h post-treatment, and were used for in vivo IP assay using anti-Pha13 antibodies. Samples after IP were analyzed by immunoblotting using anti-Pha13 antibody. (A and B) Extracted total proteins (input) stained by coomassie brilliant blue (CBB) served as a loading control.

https://doi.org/10.1371/journal.ppat.1007288.g008

The results indicate that both mock and SA treatment increased the accumulation of Pha13; however, SA treatment may induce the degradation of Pha13 through 26S proteasome activity.

In addition, in our assay using anti-ubiquitin antibody to detect the precipitated protein(s), we detected a protein band with molecular weight of around 43 kD increased with time in both SA treated-P. aphrodite and mock treated-P. aphrodite. However, increased signals of the 43 kD protein band was detected more in SA treated- than in mock treated-P. aphrodite (Fig 8A). The results indicate that SA treatment can have multiple effects on Pha13 at protein levels.

Subcellular localization of Pha13

Analysis of Pha13 amino acid sequence by the use of PredictProtein (https://www.predictprotein.org/) (Fig 1B) revealed putative nuclear localization signal. We therefore wondered whether Pha13 localized to the nucleus. To observe the subcellular localization of Pha13, we transiently expressed Pha13 fused to GFP either at the N- or C terminal, designated 35S::G-Pha13 and 35S::Pha13-G, respectively, in protoplasts isolated from P. aphrodite. Protoplasts were collected 24 h post-transfection and examined by confocal microscope. In about 50% of cells expressing G-Pha13 and Pha13-G, green fluorescence was observed exclusively in the nucleus (S5 Fig). Nucleus-specific green fluorescence was not observed in the GFP control vector.

Overexpression of Pha13 in Arabidopsis enhances resistance to viral and bacterial pathogens

Previously, phylogenetic analysis revealed that SAP proteins are evolutionarily conserved among plants [28]. To analyze whether Pha13 mediated plant immunity is conserved in plants, we transformed 35S promoter driven overexpression Pha13 (pPha13-oe) constructs into Arabidopsis (Col-0). Homozygous T3 plants derived from 3 T1 transgenic lines were selected for further disease resistance analysis. The overexpression of Pha13 was confirmed by qRT-PCR on the homozygote progenies (Fig 9A). All Pha13 overexpression transgenic Arabidopsis displayed higher shoot fresh weight and longer radius of leaf than Arabidopsis wild-type (S6A–S6C Fig) and confer an early-flowering phenotype (S6D and S6E Fig).

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Fig 9. Overexpression of Pha13 in engineered transgenic Arabidopsis enhances resistance against viruses and bacteria.

(A to C) Expression level of Pha13 (A) and accumulation level of Tobacco rattle virus (TRV) (B) and Cucumber mosaic virus (CMV) (C) were analyzed by qRT-PCR from leaves of WT (Col-0) or transgenic Arabidopsis. Data represent mean ± SD; n = 4 biological replicates; *, P < 0.05, Student’s t-test compared to vector. Actin was used as an internal control for normalization. (D) Disease symptoms of WT (Col-0) or transgenic Arabidopsis inoculated with CMV at 9 dpi. Images are one representative plant from four replicates; Scale bar, 1 cm. (E) Disease symptoms of WT (Col-0) or transgenic Arabidopsis inoculated with 1 × 10⁷ cfu/ml Pseudomonas syringae pv. tomato DC30000 (PstDC3000) at 3 dpi. Images are three representative leaves from five plants; Scale bar, 1 cm. (F) The growth analysis of PstDC3000 in the infected leaves of WT or transgenic Arabidopsis. Data represent mean ± SD; n = 5 biological replicates. Different letters indicate statistically significant differences analyzed by one-way analysis of variance (ANOVA) Tukey’s test (P < 0.05).

https://doi.org/10.1371/journal.ppat.1007288.g009

We inoculated Tobacco rattle virus (TRV), which does not cause symptoms on Col-0, by agroinfiltration to four wild-type and four Pha13 overexpressing Arabidopsis. The results revealed that TRV accumulation is dramatically decreased in all three Pha13 overexpressing transgenic lines at 9 days post-inoculation (dpi) (Fig 9B). In addition, we also mechanically inoculated Cucumber mosaic virus (CMV) to wild-type and Pha13 overexpressing Arabidopsis. The results showed that Pha13 overexpressing Arabidopsis greatly enhanced resistance to CMV (Fig 9C and 9D). In addition to viruses, we also inoculated Pseudomonas syringae pv. tomato DC30000 (PstDC3000) to five T3 progenies of At-pha13#1, At-pha13#4 and At-pha13#15. The three Arabidopsis Pha13 transgenic lines showed enhanced resistance to PstDC3000 compared to Arabidopsis wild-type at 3 dpi (Fig 9E and 9F).

AtSAP5—An Arabidopsis homolog of Pha13 is involved in viral resistance

Our phylogenetic analysis revealed that Pha13 is most related to Arabidopsis AtSAP5 (S7 Fig). Therefore, we generated transgenic Arabidopsis (Col-0) to overexpress AtSAP5, and also generated RNAi lines to express hairpin RNA of AtSAP5. The over- and down-expression of AtSAP5 were confirmed by qRT-PCR in two randomly-selected overexpression (AtSAP5-oe-4 and AtSAP-oe-11) and RNAi (AtSAP5-RNAi-3 and AtSAP5-RNAi-7) lines (Fig 10A). No obvious difference in phenotype was observed among the transgenic plants and WT (S8 Fig).

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Fig 10. The Arabidopsis homologue of Pha13, AtSAP5, is induced by SA and involved in virus resistance.

(A) The expression of AtSAP5 were analyzed by qRT-PCR in the WT, AtSAP5 overexpression lines (AtSAP5-oe-4 and AtSAP5-oe-11), and RNAi lines (AtSAP5-RNAi-3 and AtSAP5-RNAi-7). Data represent mean ± SD; n = 3 biological replicates *, p<0.05, Student’s t-test compared to WT (B) The accumulation levels of Cucumber mosaic virus (CMV) were analyzed by qRT-PCR from leaves of WT inoculated with buffer (Mock), CMV-inoculated WT, or CMV-inoculated transgenic Arabidopsis. Data represent mean ± SD; n = 5 biological replicates *, p<0.05, Student’s t-test compared to WT (C) Disease symptoms of CMV inoculated WT or transgenic Arabidopsis. The experiments were repeated twice with similar results, and 5 plants were used in each inoculation. Photo of one representative plant from each inoculation taken at 9 days post inoculation are shown here; Scale bar, 1 cm. (D) Time-course expression of AtSAP5 in SA-treated wild-type (WT, Col-0) Arabidopsis. Expression level of AtSAP5 was analyzed by qRT-PCR from leaves at 1 and 6 h post-treatment. Water treatment was used as a mock control. Data represent mean ± SD; n = 3 biological replicates *, p<0.05, Student’s t-test compared to mock. Actin was used as an internal control for normalization.

https://doi.org/10.1371/journal.ppat.1007288.g010

We mechanically inoculated CMV to WT (Col-0), AtSAP5-oe-4, AtSAP5-oe-11, AtSAP5-RNAi-3, and AtSAP5-RNAi-7. Total RNA was extracted from CMV inoculated-WT, -AtSAP5-oe-4, -AtSAP5-oe-11, -AtSAP5-RNAi-3 and -AtSAP5-RNAi-7, and used for qRT-PCR in the quantification of CMV. The results showed that CMV was detected in WT (Fig 10B). In comparison, the level of CMV was below our accurate detection limit in AtSAP5-oe-4 and AtSAP5-oe-11, whereas higher CMV accumulation was observed in AtSAP5-RNAi-3 and AtSAP5-RNAi-7 compared to WT (Fig 10B). While similar disease symptom was observed on CMV infected-WT, -AtSAP5-RNAi-3, and -AtSAP5-RNAi-7, no disease symptom on CMV-infected AtSAP5-oe-4 and AtSAP5-oe-11 were observed (Fig 10C). Our data indicates that AtSAP5 in Arabidopsis is also involved in virus resistance (Fig 10B and 10C).

AtSAP5 is involved in the expression of NPR1 and NPR1-independent genes

To analyze whether AtSAP5 also plays similar role as Pha13, total RNA was extracted from WT, AtSAP5-oe-4, AtSAP5-oe-11, AtSAP5-RNAi-3 and AtSAP5-RNAi-7, with H₂O (Mock) or SA treatment and used for the detection of AtSAP5, NPR1, PR1, RdR1 and GRXC9 expression by qRT-PCR (Fig 11).

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Fig 11. AtSAP5 is involved in the expression of NPR1 and NPR1-independent genes.

(A-D) The expression level of NPR1 (A), PR1 (B), RdR1 (C), and GRXC9 (D) were analyzed by qRT-PCR in H₂O (Mock) or SA-treated WT, AtSAP5 overexpression (AtSAP5-oe-4 and AtSAP5-oe-11) and RNAi lines (AtSAP5-RNAi-3 and AtSAP5-RNAi-7). Data represent mean ± SD; n = 3 biological replicates; *, p<0.05, Student’s t-test. The dashed lines indicate the sample with significant difference compared to WT. The colored lines indicate the significant difference was analyzed between corresponding samples treated with SA or mock. Actin was used as an internal control for normalization.

https://doi.org/10.1371/journal.ppat.1007288.g011

Our analysis also revealed that SA treatment induced the expression of AtSAP5 at 1 and 6 hours post treatment (hpt) in WT (Fig 10D). The expression level of NPR1 and RdR1 is similar in WT, AtSAP5-oe-4, AtSAP5-oe-11, AtSAP5-RNAi-3 and AtSAP5-RNAi-7 without treatment (S9 Fig). The expression of PR1 and GRXC9 is below our accurate detection limit in WT, AtSAP5-oe-4, AtSAP5-oe-11, AtSAP5-RNAi-3 and AtSAP5-RNAi-7 without any treatment (S9 Fig).

With mock (H₂O) treatment, higher expression of NPR1 and RdR1 was observed in AtSAP5-oe-4 and AtSAP-oe-11 (Fig 11A and 11C) as compared to H₂O treated-WT at 1 hpt. With SA treatment, significantly higher expression of NPR1 (1 hpt), PR1 (1 hpt), and GRXC9 (1 and 6 hpt) was observed in AtSAP5-oe-4 and AtSAP-oe-11 as compared to SA treated-WT (Fig 11A, 11B and 11D). No decreased expression of NPR1, PR1, RdR1 and GRXC9 was observed in AtSAP5-oe-4 and AtSAP-oe-11 as compared to WT regardless of the treatment or time-point (Fig 11A–11D).

With mock (H₂O) treatment, decreased expression of PR1 was observed in both AtSAP5-RNAi-3 and AtSAP5-RNAi-7 as compared to H₂O treated-WT at 6 hpt (Fig 11B). With SA treatment, decreased expression of NPR1 (6 hpt), PR1 (1 and 6 hpt) and GRXC9 (1 hpt) was observed in both AtSAP5-RNAi-3 and AtSAP5-RNAi-7, and decreased RdR1 (1 and 6 hpt) expression was observed in AtSAP5-RNAi-7 as compared to SA treated-WT (Fig 11A–11D). No increased expression of NPR1, PR1, RdR1 and GRXC9 were observed in AtSAP5-RNAi-3 and AtSAP5-RNAi-7 as compared to WT regardless of the treatment or time point (Fig 11A–11D).

Collectively, our data using transgenic Arabidopsis overexpressing or silencing AtSAP5 suggests that AtSAP5 is involved in the expression of NPR1 and NPR1-independent genes.

Subject materials and growth conditions

The commercial orchid variety, Phalaenopsis aphrodite var. Formosa, was purchased from Taiwan Sugar Research Institute (Tainan, Taiwan). P. aphrodite, P. equestris and transgenic P. equestris (35S∷FLAG-Pha13) plants were all first tested for two prevalent orchid viruses, Odontoglossum ringspot virus (ORSV) and CymMV, as detected by RT-PCR with primer pairs, ORSV-F/ORSV-R and CymMV-F/CymMV-R (S3 Table), before maintaining in a greenhouse with a controlled 12-h photoperiod (200 μmol m^-2s^-2) at 25°C/25°C (day/night). The Arabidopsis WT (Col-0) and all transgenic Arabidopsis were maintained in a greenhouse with a controlled 12-h photoperiod (200 μmol m^-2s^-2) at 22°C/22°C (day/night) for ~four weeks before analysis. Cucumber mosaic virus isolate 20 was maintained in the Arabidopsis thaliana (Col-0) as inoculation source in our study. The infectious clones of Cymbidium mosaic virus (pCambia-CymMV) and Tobacco rattle virus (pTRV1 and pTRV2) were transformed into Agrobacterium tumefaciens C58C1 (pTiB6S3ΔT)^H (described below) for inoculation of Orchid or Arabidopsis through agroinfiltration. E. coli strains BL21 was grown on Luria broth (LB) agar plates or in LB broth. Pseudomonas syringae pv. tomato DC30000 was grown in the King’s B medium (20 g/L proteose peptone, 1.5 g/L K₂HPO4, 10 ml glycerol, and 1.5 g/L MgSO4 •7H₂O, pH 7.0). Saccharomyces cerevisiae strain AH109 was grown on the YPAD (Yeast extract 10 g/L, Peptone 20 g/L, Dextrose 20 g/L, and Adenine sulfate 0.4 g/L) agar plate or YPAD broth.

Phytohormone treatment

Sodium salicylate (50 mM) (Sigma), methyl jasmonate (45 μM) (Sigma), and aminocyclopropanecarboxylic acid (660 μM) (Sigma) were directly rubbed on leaves of P. aphrodite by cotton swab. Leaf samples were collected at 0 h, 1 h, 3 h, 6 h, 12 h, 24 h, 48 h and 72 h after treatment. For Arabidopsis, 1 mM SA was sprayed on the leaves, and samples were collected at 1 h, 6 h, and 24 h after treatment.

Sequence analysis

The protein domains of Pha13 were analyzed by use of PROSITE database of ExPASy Proteomics Server (http://ca.expasy.org/) and Conserved Domain Database of NCBI database (http://www.ncbi.nlm.nih.gov/). The predicated nuclear localization signal of Pha13 was analyzed by use of PredictProtein (https://www.predictprotein.org/)

Phylogenetic tree analysis

Phylogenetic analysis of Pha13 was conducted with 12 orchid A20/AN1 domain containing stress associated proteins (SAPs) and previously characterized 14 SAPs from Arabidopsis thaliana and 18 SAPs from Oryza sativa. The sequence alignment was performed by use of the Clustal W algorithm of the DNASTAR MegAlign software (DNASTAR, WI, USA). An unrooted phylogenetic tree was constructed using the neighbor-joining method by MEGA5 with 1000 bootstrap replicates. The sequences of SAPs from orchids, A. thaliana, and O. sativa were obtained from the websites, Orchidstra database (http://orchidstra2.abrc.sinica.edu.tw/), TAIR (http://www.arabidopsis.org) and the Rice Genome Annotation Project (http://rice.plantbiology.msu.edu), respectively. The accession of each gene used for analysis is indicated in S7 Fig.

RNA isolation, semi-quantitative and qRT-PCR analysis

Total RNA was extracted as described previously [27]. For semi-quantitative RT-PCR, 1 μg of total RNA and oligo (dT) primer were used to synthesize the cDNA. The PCR was performed using the gene-specific primer pairs (S3 Table). The results of semi-quantitative RT-PCR were analyzed by ImageJ software for the relative quantification. For qRT-PCR, cDNA was synthesized from 500 ng of DNA-free RNA and oligo (dT) by use of PrimeScript RT Reagent Kit (Perfect Real Time) (Takara Bio) following the manufacturer’s instructions (Takara Bio). The cDNA template was used for qPCR by use of SYBR Premix EX Taq II (Ti RNase H Plus) Kit (Takara Bio) in an ABI Prism 7500 sequence detection system (Applied Biosystems). The PhaUbiquitin 10 or Actin was used as an internal quantification control. The primer pairs used in this study are listed in (S3 Table).

Construction of transient silencing and virus-induced gene-silencing vectors

For construction of transient silencing vector of Pha13, the oligonucleotide pair Pha13-hpRNA-F/Pha13-hpRNA-R (S3 Table) was used to generate the hairpin dsDNA fragments. The hairpin dsDNA fragments were cloned into the Gateway entry vector pENTR/D-TOPO (Thermo Fisher-Scientific) following the manufacturer’s instructions to generate pENTR-Pha13-hpRNA. Then, LR Gateway cloning reaction (Thermo Fisher-Scientific) was conducted to transfer the hairpin RNA fragments from pENTR-Pha13-hpRNA into 35S promoter driven pB7GWIWG2(I) [48] to obtain phpPha13. The method for construction of transient silencing vector of PhaNPR1, PhaRdR1, and PhaGRX was similar to that described above, except the oligonucleotide pairs PhaNPR1-hpRNA-F/PhaNPR1-hpRNA-R, PhaRdR1 -hpRNA-F/PhaRdR1-hpRNA-R, and PhaGRX-hpRNA-F/ PhaGRX-hpRNA-R (S3 Table) were used to generate the hairpin dsDNA fragments.

For construction of virus-induced gene-silencing vector, plant RNA was used as a template to amplify the fragments of Pha13 by RT-PCR with the primer pair attB1-Pha13-F/attB2-Pha13-R (S3 Table). The amplified fragments were cloned into the pCambia-CymMV-Gateway vector [27] by the BP Clonase II enzyme mix (Thermo Fisher-Scientific) to generate pCambia-CymMV-Pha13.

Construction of Pha13 overexpression vector

For construction of Pha13 transient overexpression vector, plant total RNA was used as a template to amplify the N terminal FLAG tagged of Pha13 by RT-PCR with the primer pairs FLAG-Pha13ORF-F/Pha13ORF-R (S3 Table). The FLAG-Pha13 fragments were cloned into the Gateway entry vector pENTR/D-TOPO (Thermo Fisher-Scientific) following the manufacturer’s recommendations to generate pENTR-FLAG-Pha13. Then, LR Gateway cloning reaction (Thermo Fisher-Scientific) was used to transfer the FLAG-Pha13 fragments from pENTR-FLAG-Pha13 into the 35S promoter driven overexpression vector, pK2GW7 [48], to obtain pPha13-oe. For generation of A20 and/or AN1 mutant on pPha13-oe (Fig 1C and 1D), site-directed mutagenesis was conducted by QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies). For A20 mutant, we substituted the conserved 3^rd and 4^th cysteine to glycine at A20 (C29G and C32G). For AN1 mutant, we substituted the conserved 3^rd cysteine and 1^st histidine to glycine at AN1 (C111G and H121G). The A20 mutated clone and AN1 mutated clone was designated pPha13A20m and pPha13AN1m, respectively. The A20 and AN1 double mutated clones was designated pPha13A20mAN1m. Primer pairs used for site directed mutagenesis are listed in S3 Table.

Transgenic Phalaenopsis orchid and Arabidopsis plants

For construction of overexpression vector to generate transgenic Phalaenopsis orchid, the FLAG-Pha13 fragment was transferred from pENTR-FLAG-Pha13 (described above) into binary vector, pH2GW7 [48], to obtain pHPha13. pHPha13 was used to generate transgenic P. equestris orchid using the method described by Hsing et al. [49]. For the construction of overexpression and RNAi vector of AtSAP5, pAtSAP5-oe or phpAtSAP5-RNAi, we followed similar method as described above for the construction of pPha13-oe and phpPha13, except the primers used to amplify the full length and the fragment of AtSAP5 were AtSAP5ORF-F/AtSAP5ORF-HA-R (for overexpression vector, pAtSAP5-oe) and AtSAP5-RNAi-F/AtSAP5-RNAi-R (for RNAi vector, phpAtSAP5-RNAi; S3 Table).

For the generation of transgenic Arabidopsis, the plants were transformed using the floral dip method with Agrobacterium tumefaciens strain GV3101 carrying the pPha13-oe, pAtSAP5-oe, or phpAtSAP5-RNAi to generate the transgenic plants.

Agroinfiltration

Agroinfiltration on orchid was conducted as previously described [27], with modification. pCambia-CymMV, pB7GWIWG2, pK2GW7 and their derivatives and were transformed into Agrobacterium tumefaciens C58C1 (pTiB6S3ΔT)^H by electroporation. Briefly, A. tumefaciens strains were incubated at 28°C until reaching an OD₆₀₀ to 1.0. After centrifugation, 20 ml AB-MES medium (17.2 mM K₂HPO₄, 8.3 mM NaH₂PO₄, 18.7 mM NH₄Cl, 2 mM KCl, 1.25 mM MgSO₄, 100 μM CaCl₂, 10 μM FeSO4, 50 mM MES, 2% glucose (w/v), pH 5.5) with 200 μm acetosyringone [50] was used to re-suspend the cells. After culturing overnight, 2 ml of infiltration medium containing 50% MS medium (1/2 MS salt supplemented with 0.5% sucrose (w/v), pH 5.5), 50% AB-MES and 200 μm acetosyringone [50] were used for infiltration.

For the agroinfiltration on Arabidopsis, same method was used as described above except pTRV1 and pTRV2 were individually transformed into A. tumefaciens C58C1 (pTiB6S3ΔT)^H by electroporation, and overnight culture of A. tumefaciens containing pTRV1 and pTRV2 was adjusted to OD₆₀₀ of 0.5 and mixed at 1:1 ratio prior to infiltration.

Cymbidium mosaic virus (CymMV), Cucumber mosaic virus (CMV), and Tobacco rattle virus (TRV) inoculation and accumulation assay

To assay the effect of Pha13, PhaRdR1, or PhaGRX in CymMV accumulation, we first inoculated CymMV in P. aphrodite. For inoculation of CymMV, agroinfiltration (described above) was performed to infiltrate A. tumefaciens carrying pCambia-CymMV in the leaf tip of P. aphrodite. The CymMV-infected P. aphrodite were maintained at least 14 days before further analysis. To assay the effect of transient silencing (Pha13, PhaRdR1, or PhaGRX) or transient overexpression (Pha13, or their derived mutants), A. tumefaciens carrying the control vector pB7GWIWG2 (for silencing), pK2GW7 (for overexpression), silencing vectors or overexpression vectors (described above) were infiltrated into the leaves. After agroinfiltration, a pair of disks (6 mm diameter) were immediately (defined as 0 dpi) collected from both the control and assay vector infiltrated regions. After 5 dpi, another pair of disks were collected from the same infiltrated region. Total RNA extracted from the samples was used as a template to analyze the accumulation of CymMV by use of qRT-PCR. The ratio of CymMV accumulation at 0 dpi to 5 dpi was calculated for relative quantification.

For TRV inoculation, agroinfiltration (as described above) was performed by infiltrating A. tumefaciens carrying pTRV1 or pTRV2 (1:1) to three leaves of four-week-old Arabidopsis by use of syringe. After 9 dpi, total three disks from three different distal leave were collected and quantified the accumulation of TRV by use of qRT-PCR.

For inoculation with CMV, CMV-infected Arabidopsis leaves were ground with 0.01 M potassium phosphate buffer by mortar and pestle for use as the inoculation source. Four-week-old Arabidopsis leaves were inoculated mechanically (pre-dusted with 300-mesh Carborundum) with the CMV inoculation source. After 9 dpi, three disks from three different distal leave were collected and CMV accumulation was analyzed by use of qRT-PCR.

Microarray analysis

Total RNA was extracted from leaves of P. aphrodite infiltrated with agrobacterium carrying vector (pK2GW7) or pPha13-oe 5 days after infiltration. For microarray, 0.2 μg of total RNA was amplified by a Low Input Quick-Amp Labeling kit (Agilent Technologies) and labeled with Cy3 (CyDye, Agilent Technologies, USA) during the in vitro transcription process. An amount of 0.6 μg of Cy3-labled cRNA was fragmented at 60°C for 30 minutes. Corresponding fragmented labeled cRNA was then pooled and hybridized to Agilent P. aphrodite 8 × 60K Microarray (Agilent design ID: 033620) [51] at 65°C for 17 hours. After washing and drying steps, the microarrays were scanned with an Agilent microarray scanner (Agilent Technologies) at 535 nm for Cy3. The array image was analyzed by Feature Extraction software version 10.7.1.1 using the default setting. For the microarray analysis, data were analyzed from 3 biological repeats using GeneSpring (Agilent Technologies, http://www.agilent.com). Pha13-responsive differentially expressed genes (DEGs) were identified based on significance compared to vector control (unpaired t test P < 0.05). Microarray data was deposited in the public repository GEO database (https://www.ncbi.nlm.nih.gov/geo/) with accession number GSE93248.

Gene ontology analysis

Gene ontology (GO) classification was conducted with all the Pha13 -responsive DEGs by use of GO enrichment analysis algorithm of gene ontology database (http://geneontology.org/). All the Pha13-responsive DEGs were categorized into subcategories of biological process GO terms.

Isolation and detection of Pha13 and Pha13 A20 and/or AN1 domain mutant protein

Total proteins were extracted from leaves of transgenic orchid or leaves of orchids infiltrated with agrobacterium carrying vector (pk2GW7), overexpression clones of wild-type Pha13 (pPha13-oe), or the respective A20 and/or AN1 mutant clone (pPha13A20m, pPha13AN1m or pPha13A20mAN1m) as previously described [52] with some modification. The boiled extraction buffer (4 M urea, 5% SDS, 15% glycerol, 100 mM Tris-HCl, pH 8, with freshly added 2 mM phenylmethylsulfonyl fluoride, 2 mg mL-1 and 1X complete protease inhibitor [Roche]) was used to extract the total proteins. The Pha13 or Pha13 A20 and/or AN1 domain mutant protein was analyzed by immunoblotting with antibody against Pha13 followed by HRP conjugated anti-rabbit antibodies (Abcam). The protein level of tubulin (loading control) was analyzed by immunoblotting with antibody against tubulin followed by HRP conjugated goat anti-mouse antibodies (GE Healthcare Life Sciences).

Construction, expression, and purification of recombinant proteins

Full-length Pha13, or mutant cDNA were amplified by PCR with primer pairs, NdeI-Pha13-F/NotI-Pha13-R (S3 Table), with previously described Pha13 overexpression or mutated clones as templates. PCR amplified gene fragments were cloned into the pET24b expression vector (Novagen) with fused C-terminal histidine tag (His-tag) to produce protein expression plasmids, pETPha13, pETPha13A20m, pETPha13AN1m and pETPha13A20mAN1m. The constructed plasmids were transformed into Escherichia coli strain BL21 (DE3) for protein expression. Bacteria were cultured at 37°C to an OD₆₀₀ of 0.5 and transferred to 25°C for 1.5 hours for Pha13, Pha13A20m, Pha13AN1m, and Pha13A20mAN1m protein induction. Protein induction was performed by addition of isopropylthio-β-galactoside (IPTG; Sigma) to a final concentration of 1 mM. His-tagged recombinant protein was purified by TALON Superflow (GE Healthcare Life Sciences) according to the manufacturer’s description. The elution was carried out with 250 mM imidazole (Sigma).

E3 ubiquitin ligase activity assay

In vitro ubiquitination assays were performed as described [33] with modification. An amount of 3 μg purified His-tagged recombinant proteins (described above) were used for each ubiquitination reaction. Reactions were incubated at 30°C for 3 hours and analyzed by SDS-PAGE followed by immunoblot analysis. Blots were probed using anti-FLAG antibodies (Sigma) followed by HRP conjugated goat anti-mouse antibodies (GE Healthcare Life Sciences).

Ubiquitin-binding assay

Full-length AtSAP5 cDNA was amplified by PCR with primer pairs, NdeI-AtSAP5-F/XhoI-AtSAP5-R (S3 Table), and were cloned into pET24b following the same approach as the cloning of pETPha13 described above to generate the pETAtSAP5. His-tagged AtSAP5, Pha13 and Pha13A20m recombinant proteins were expressed by E. coli and purified with affinity resin. Purified recombinant proteins (60 μg) were immobilized on magnetic beads (60 μl) using Mag-beads Carboxyl Labeling kit (Toolsbiotech) following the manufacturer’s instructions. Recombinant proteins (6 μl) conjugated magnetic beads were incubated with 4 μg of linear, K6, K11, K29, K33, K63-inked tetraubiquitin chains or K48-linked polyubiquitin (Ub3-Ub7) chains (BostonBiochem) for 18 h at 4°C in pull-down buffer (20 mM Tris–HCl pH 7.5, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 10 mM ZnSO₄, 0.5 mM DTT). After pull-down, the beads were washed three times with pull-down buffer. The pull-down proteins were boiled with SDS-sample buffer and analyzed by SDS-PAGE and immunoblotting with anti-ubiquitin antibody (Research and Diagnostic Systems).

Immunoprecipitation (IP)

The IP assay for Pha13 was performed using total proteins extracted from 0.25 g of Phalaenopsis orchid leaves with 200 ul immunoprecipitation buffer (50 mM Tris-pH 7.5, 0.1% NP40, 10 mM MgCl₂, 150 mM NaCl, 10 uM MG132, 1X complete protease inhibitor [Roche]). The extract was centrifuged at 12000 ×g for 10 min at 4 °C. Then, the supernatants were transferred to the non-stick tube, and pre-cleared with protein A sepharose beads (GE Healthcare Life Sciences) 1 hours at 4 °C with gentle shaking. After, the extract was centrifuged at 1500 ×g for 2 min at 4 °C, the supernatant was further incubated with the anti-Pha13 antibodies for 2 hours at 4 °C with gentle shaking, followed by incubation with protein A sepharose beads (GE Healthcare Life Sciences) for 2 hours at 4 °C with gentle shaking. The beads were washed three times with ice-cold immunoprecipitation buffer and eluted by SDS-PAGE sampling buffer. The eluted proteins were analyzed by immunoblotting using the anti-Pha13 antibody and anti-ubiquitin antibody (Research and Diagnostic Systems).

Protein degradation assays

For the degradation assay of Pha13, the leaves of P. aphrodite were treated with H₂O (Mock) or SA, and immediately followed by infiltration of DMSO or 40 uM MG132. Total proteins were extracted from the treated samples at 3 hours post-treatment and followed by the IP assay using anti-Pha13 antibodies (described above). The eluted proteins were analyzed by immunoblotting using the anti-Pha13 antibody.

Preparation and transfection of protoplasts

For construction of vectors used for subcellular localization analysis, the primer pairs, Pha13-ORF-F/Pha13-ORF-R and Pha13-ORF-F/Pha13-ORF-NONSTOP-R (S3 Table) were used to amplify 2 sets of Pha13 ORF (with or without stop codon). All the amplified ORF fragments of Pha13 were cloned into the Gateway entry vector pCR 8/GW/TOPO Gateway (Thermo Fisher-Scientific) following the manufacturer’s recommendations to generate pCR8-Pha13 and pCR8-Pha13-NONSTOP. Then, LR Gateway cloning reaction (Thermo Fisher-Scientific) was used to transfer the ORF fragment of Pha13 from pCR8-Pha13 into p2FGW7 driven by 35S promoter [48] to obtain N-terminal GFP fused clones (pG-Pha13). To obtain C-terminal GFP-fused clones (pPha13-G), we transferred pCR8-Pha13-NONSTOP into p2GWF7. Protoplast isolation and transfection were as described [27]. Transformed protoplasts were detected for florescence signals by confocal microscopy (Zeiss LSM 780, plus ELYRA S.1) with excitation at 488 nm and emission at 500 to 587 nm for GFP, and excitation at 543 nm and emission at 600 to 630 nm for mCherry.

Yeast two-hybrid screening

Full-length Pha13 cDNA was amplified by PCR with primer pairs, NdeI-Pha13-F/EcoRI-Pha13-R (S3 Table). The fragments were cloned into the pGBK vector (Clontech) with fused N-terminal GAL4 DNA binding domain to produce pGBKPha13 plasmid as a bait vector. pGBKPha13 was transformed into AH109 yeast strain as competent cells for yeast two-hybrid cDNA library screening. Total RNA isolated from Phalaenopsis leaf tissue (24 hours after SA treatment) was used for cDNA library construction. The cDNA library was generated with a Make Your Own “Mate & Plate” Library System (Clontech) following the user manual. Candidate yeast colonies were picked up PCR amplification with primer pairs, T7promoter/3’ ADprimer (Clontech). Amplified DNA fragments were sequenced, and the sequences were used in a blast search on an orchid database (http://orchidstra2.abrc.sinica.edu.tw/) to identify corresponding genes. Full lengths of candidate genes were amplified with RT-PCR (S3 Table) and cloned into pGAD vector for further confirmation analysis. Yeast strains containing the appropriate bait and prey plasmids were cultured in liquid 2-dropout medium (leucine^- and tryptophan^-) overnight. The overnight yeast culture was diluted to an OD₆₀₀ of 0.06 and spotted on selection plates (containing histidine^-, leucine^-, tryptophan^- and 5-bromo-4-chloro-3-indolyl-alpha-D-galactopyranoside) for growth assay. Fragments of different N-terminus deletions of Pha13 were generated with restriction enzyme digestions of FseI (NEB), ApaI (NEB), SgrAI (NEB), BtgI (NEB), or PspXI (NEB). Fragments of C-terminus deletions were generated by PCR amplification using primer pairs, Nd13Fs412F/EcoRI-Pha13-R, Nd13Ap361F/EcoRI-Pha13-R, Nd13Sg247F/ EcoRI-Pha13-R, Nd13Bt136F/EcoRI-Pha13-R, and Nd13Ps88F/EcoRI-Pha13-R (S3 Table). Pha13 A20 domain was amplified by PCR with primer pairs, Nd13A20F/BH13A20R (S3 Table). Pha13 AN1 domain was amplified with primer pairs, Nd13AN1F/BH13AN1R (S3 Table). The yeast two-hybrid assay of the truncated Pha13 fragments were conducted as above.

Pseudomonas syringae pv. tomato DC30000 (PstDC3000) inoculation and quantification assay

Four-week-old Arabidopsis were dipped in liquid suspension of 10⁷ cfu/mL PstDC3000 in 10 mM MgSO4 containing 0.01% Silwet L-77 (Lehle Seeds) for 5 min. After inoculation, plants were kept at 100% relative humidity. For bacterial population quantification, three discs were collected from individual inoculated-plants after 3 days post-inoculation and grounded in sterile water. Serial dilution was performed and the King’s B medium containing 100 ug/ml rifampicin for colony counting.

Quantification and statistical analysis

Data are presented as mean ± SD. The pair-wise t-test was performed to analyze the statistical significance between samples. The one-way analysis of variance (ANOVA) followed by Tukey’s test was performed to analyze the statistical significance for data of Pha13 expression level in different tissues and bacterial pathogen growth. In transient overexpression analysis, three plant replicates with expression level of at least 40% increase in Pha13 or the respective A20 and/or AN1 mutant, compared to vector control, were used for statistical analysis.

Accession numbers

Pha13 (PATC148746), PhaPR1 (PATC126136), PhaNPR1 (PATC135791), PhaRdR1; (PATC143146), PhaGRX (PATC068819), PhaUBQ10 (PATC230548), PhaJAZ1 (PATC141437), PhaACO2 (PATC139319), AtActin (At3G18780), AtSAP5 (AT3G12630), OsSAP3 (LOC_Os01g56040.1), OsSAP5 (LOC_Os02g32840.1).