The heat shock transcription factor PsHSF1 of Phytophthora sojae is required for oxidative stress tolerance and detoxifying the plant oxidative burst.

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Environmental Microbiology (2015) 17(4), 1351–1364

doi:10.1111/1462-2920.12609

The heat shock transcription factor PsHSF1 of Phytophthora sojae is required for oxidative stress tolerance and detoxifying the plant oxidative burst

Yuting Sheng,1 Yonglin Wang,1 Harold J. G. Meijer,2 Xinyu Yang,1 Chenlei Hua,2 Wenwu Ye,1 Kai Tao,1 Xiaoyun Liu,1 Francine Govers2,3 and Yuanchao Wang1* 1 Department of Plant Pathology, Nanjing Agricultural University, Nanjing 210095, China. 2 Laboratory of Phytopathology, Wageningen University, Droevendaalsesteeg 1, Wageningen NL-6708 PB, The Netherlands. 3 Centre for BioSystems Genomics (CBSG), P.O. Box 98, Wageningen 6700 AB, The Netherlands. Summary In the interaction between plant and microbial pathogens, reactive oxygen species (ROS) rapidly accumulate upon pathogen recognition at the infection site and play a central role in plant defence. However, the mechanisms that plant pathogens use to counteract ROS are still poorly understood especially in oomycetes, filamentous organisms that evolved independently from fungi. ROS detoxification depends on transcription factors (TFs) that are highly conserved in fungi but much less conserved in oomycetes. In this study, we identified the TF PsHSF1 that acts as a modulator of the oxidative stress response in the soybean stem and root rot pathogen Phytophthora sojae. We found that PsHSF1 is critical for pathogenicity in P. sojae by detoxifying the plant oxidative burst. ROS produced in plant defence can be detoxified by extracellular peroxidases and laccases which might be regulated by PsHSF1. Our study extends the understanding of ROS detoxification mechanism mediated by a heat shock TF in oomycetes. Introduction Plants have developed complex but efficient defence systems to counteract plant pathogenic bacteria, fungi, nematodes and oomycetes during invasion, colonization Received 20 May, 2014; accepted 20 August, 2014. *For correspondence. E-mail [email protected]; Tel. +(86) 25 84399071; Fax (+86) 25 84395325.

© 2014 Society for Applied Microbiology and John Wiley & Sons Ltd

and spreading (Tyler, 2002; De Gara et al., 2003). One of the major ubiquitous and earliest responses in plant defence is the rapid accumulation of reactive oxygen species (ROS) upon pathogen recognition at the plant surface (Apostol et al., 1989). The apoplastic production of ROS is catalysed by plasma membrane-localized nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) (Doke et al., 1996). Besides the deleterious direct effect on pathogens such as damaging DNA, proteins, lipids and other cell components, apoplastic ROS production might serve additional purposes such as the activation of plant defence responses through programmed cell death (Greenberg, 1997). It also acts as a secondary messenger in the signal transduction pathway of the pathogen-associated molecular pattern-triggered immunity (Wojtaszek, 1997; Neill et al., 2002). Pathogens have adapted to plant-derived ROS by the development of detoxification systems in order to infect successfully (Apel and Hirt, 2004). Antioxidants such as ascorbate, glutathione (GSH), tocopherol, flavonoids, alkaloids and carotenoids are produced as a non-enzymatic strategy. Enzymatic scavengers of ROS scavenging include superoxide dismutases, ascorbate peroxidases, cytochrome c peroxidases, GSH peroxidases and catalases (Apel and Hirt, 2004). In fungi, the expression of genes encoding ROS detoxification proteins is highly dependent on transcription factors (TFs) (Molina and Kahmann, 2007; Lin et al., 2009). In the rice blast fungus Magnaporthe oryzae, disruption of the basic leucine zipper (bZIP) TF gene MoAP1 results in hypersensitivity to oxidative stress and reduction of pathogenicity. Gene expression analysis showed that MoAP1 regulates pathogenicity-associated genes and redox homeostasis-related genes suggesting that this bZIP TF is essential for resistance to the oxidative damage (Guo et al., 2011). Similar studies on the bZIP TF YAP1 in Alternaria alternata (Lin et al., 2009), Candida albicans (Zhang et al., 2000) and Ustilago maydis (Molina and Kahmann, 2007) revealed that yap1 mutants displayed higher sensitivity to H2O2 and reduced pathogenicity. In Saccharomyces cerevisiae, the TFs SKN7, MSN2/4 and HSF1 are important for oxidative stress tolerance (Moye-Rowley, 2003). In the oomycete

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plant pathogen Phytophthora infestans, bZIP TFs with novel DNA-binding domains were shown to be involved in defence against oxidative stress (Gamboa-Meléndez et al., 2013) and in Phytophthora sojae, several infectionassociated bZIPs displayed oxidative stress response (Ye et al., 2013). Heat shock transcription factors (HSFs) are stressresponsive regulators conserved from yeast to humans which are the final integrators of signal cascades initiated by heat stress, chemical stimuli, developmental processes and oxidative stress (Jedlicka et al., 1997; Nover et al., 2001). In yeast, HSF1 is essential for survival (Moye-Rowley, 2003), and in Drosophila, HSF1 is important for early development (Jedlicka et al., 1997). During oxidative stress Drosophila HSF1 forms homotrimers leading to high-affinity DNA binding resulting in modulation of gene expression (Zhong et al., 1998). Also, the mouse HSF1 is required to sustain redox homeostasis and to regulate anti-oxidative defences (Yan et al., 2002). In plants, HSFs play a role in oxidative and heavy metals stress responses, biotic stress, and during development and differentiation (von Koskull-Döring et al., 2007). Oomycetes, also known as water moulds, are funguslike eukaryotic microbes which are phylogenetically distinct from fungi. They comprise plant and animal pathogens responsible for many economically important diseases. The genus Phytophthora contains over 100 species, nearly all are destructive plant pathogen that cause severe damage to a wide range of plants including many crop species (Kroon et al., 2012). Phytophthora sojae is one of the most devastating pathogens on soybean, causing damping off and root and stem rot. Losses are estimated at $1–2 billion per year worldwide (Tyler et al., 2006). The vegetative and infectious propagules of P. sojae are sporangia and zoospores. Soon after zoospores touch the plant surface, they transform into adhesive cysts that start to germinate. After ∼ 30 min, the germ tube penetrates the host tissue usually between two adjacent root epidermal cells. After ∼ 10 h, the hyphae spread into the endodermis (Enkerli et al., 1997). ROS accumulation is observed in both compatible and incompatible interactions. However, in a compatible interaction, ROS is transiently upregulated at around 3 h after inoculation, while during an incompatible interaction, ROS production is biphasic with an early response comparable to the compatible interaction and a second phase of ROS production after 8 h (Chen et al., 2008). The aim of this study was to learn more about ROS resistance in P. sojae. As a first step, we screened the genome sequence for genes encoding TFs. We specifically focused on potential ROS tolerance-related TFs and identified a large family of HSFs. Expression of one of the

HSFs was strongly correlated with ROS stress, and therefore this one, named PsHSF1, was further investigated. We show that PsHSF1 is required for oxidative stress tolerance and pathogenicity and that it functions upstream of extracellular enzymes such as peroxidases and laccases. Moreover, silencing of one of the potential targets of PsHSF1, i.e. the laccase gene PsLAC4, also resulted in reduced pathogenicity.

Results Identification and annotation of putative ROS-related TFs in Phytophthora genomes For the genome-wide screening aimed at finding candidate P. sojae TF genes involved with ROS regulation, we employed as baits yeast TF sequences such as YAP1, MSN2/4, SKN7 and HSF1 that are conserved in a wide range of organisms (Cuéllar-Cruz et al., 2008). With e-value of 1.0E−5, no homologues of YAP1 and MSN2/4 were detected in Phytophthora species. However, 22 putative P. sojae candidate HSF-encoding genes were identified that all encode proteins containing HSF-type DNA-binding domain (IPR000232). They shared relative equal similarity with yeast HSF1 and SKN7, both of which contain a HSF-type DNA-binding domain. However, all 22 genes lack a signal receiver domain (REC-domain) which is required for SKN7 function as a regulator in a two-component signalling system, so they were identified as HSFs. For all 22, corresponding orthologues were identified in the genomes of P. infestans (Haas et al., 2009) and Phytophthora ramorum (Tyler et al., 2006) (Table S1). In P. infestans, 24 putative HSF genes were found, including six without an automatically generated gene model (PiHSF-A to PiHSF-F) and in P. ramorum 18. From the 22 putative P. sojae HSF genes, three are most likely pseudogenes (Ps142187, Ps142189 and Ps142191) and two (Ps142188 and Ps142190) are nearly identical with a sequence similarity of up to 99% at the DNA level. In all 22 PsHSFs, the hallmark HSF-type DNA-binding domain is located at the N-terminus within the first 190 amino acids. In some of the PsHSFs, oligomerization domains (HA/B), a nuclear export signal (NES) and a nuclear localization sequence (NLS) were detected (Table S1). Genome analysis further revealed that several PsHSF genes are clustered in the genome (Fig. S2). This suggests that the relatively large number of HSFs in P. sojae could be the result of gene duplication. Moreover, a phylogram of HSFs from three Phytophthora species showed obvious clustering of orthologues in clades (Fig. 1A) pointing to an ancient origin of the large HSF family that has been conserved since the last common ancestor and prior to speciation.

© 2014 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology, 17, 1351–1364

PsHSF1, a ROS response regulated in Phytophthora sojae

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Fig. 1. Phylogenetic analysis of HSF proteins in Phytophthora species and HSF1 in a variety of eukaryotes. A. Phylogram of HSF proteins from P. sojae, P. ramorum and P. infestans based on neighbour-joining analysis. B. Phylogenetic analysis of HSF1 in species of fungi, plants, animals, oomycetes and diatom. Full-length amino acid sequences were used to generate the tree with MEGA 5.0. The corresponding NCBI accession numbers are Homo sapiens (NP_005517), Mus musculus (NP_032322), Drosophila melanogaster (NP_476575), Arabidopsis thaliana (NP_186949), Nicotiana tabacum (BAA83711), Oryza sativa (NP_001051938), Saccharomyces cerevisiae (NP_011442), Cryptococcus gattii (XP_003192457), Ustilago maydis (XP_761421), Candida albicans (XP_716869), Aspergillus nidulans (XP_681304), Neurospora crassa (XP_963162), Magnaporthe oryzae (XP_360699.2), Hyaloperonospora parasitica [WING_Hp0008 (605320)] (http://ftfd.snu.ac.kr/index.php?a=view), Pythium ultimum (PYU1_T007502) (http://pythium.plantbiology.msu.edu/), P. sojae (ACJ09357.1, Ps142192), P. ramorum (ACN76438.1, PrHSF1), P. infestans (XP_002905429.1, PITG_04694), Thalassiosira pseudonana (XP_002293508.1), Ectocarpus siliculosus (CBN80163.1).

PsHSF1 encodes a HSF that is induced by oxidative stress To determine the correlation between the transcription of PsHSFs and oxidative stress, we monitored the transcript levels of all PsHSF genes in P. sojae hyphae exposed to 5 mM H2O2. This mimics oxidative stress levels encountered during host invasion (Chen et al., 2008). The expression of all PsHSF genes was measured in mycelium 5, 15 and 60 min after adding H2O2. Of the 22 genes, only Ps142192, which is clustered with other HSF genes on scaffold_109 (Fig. S2), showed an expression pattern that could point to a role in responses against oxidative stress. This gene was not expressed in untreated hyphae or at a very low level, but its transcription was strongly activated within 5 min after H2O2 treatment. The 26-fold increase in transcript level rapidly reduced to around 16-fold after 15 min. Such a transient and strong induction was not observed with any of the other 21 PsHSF genes (Fig. S3). Ps142192 displays the highest similarity with the yeast TFs SKN7 (38% amino acid identity at positions 174–237) and HSF1 (44% amino acid identity at positions 66–133), although this is

solely based on the HSF-type DNA-binding domain. Since Ps142192 lacks the signal receiver domain (REC) present in SKN7, it was annotated as PsHSF1 (GenBank Accession No. FJ349602). A phylogram comprising HSFs from representative animals, fungi, plants, diatoms and other oomycetes and based on HSF sequences sharing the highest similarity with PsHSF1 revealed that the oomycete HSF1s cluster in one clade is distinct from other eukaryotes (Fig. 1B). PsHSF1 is induced by various stress conditions and during cyst germination and early infection stages PsHSF1 transcription was highly upregulated during H2O2 treatment (Fig. S3). Considering that H2O2 is relatively unstable, t-butyl hydrogen peroxide (t-BHP) was applied as alternative for mimicking ROS stress during infection at 25°C. With 0.5 mM t-BHP PsHSF1 transcript level transiently increased 71-fold within 5 min (Fig. 2A) and this confirmed that PsHSF1 expression is a response to ROS detection. Considering the high sensitivity of quantitative reverse transcript real-time polymerase chain reaction (qRT-PCR), semi-quantitative real-time polymerase chain


1354 Y. Sheng et al. stress lead to in a gradual increase in PsHSF1 transcript levels over time. Analysis of PsHSF1 expression during asexual development and infection (Fig. 2C) revealed low expression levels in mycelium and sporangia but strongly increased transcript levels in cysts and germinating cysts. This result was confirmed by SqRT-PCR (Fig. S4B). During infection, PsHSF1 was highly expressed at 1.5 h and 3 h after zoospore inoculation, but thereafter, the transcript level of PsHSF1 decreased over time (Fig. 2C). This expression pattern suggests that PsHSF1 plays a role during cyst and germinating cyst stages and early infection stages. PsHSF1 is essential for oxidative stress and heat shock stress tolerance

Fig. 2. Transcript levels of PsHSF1 under oxidative and heat shock stress and during life stages. A. Transcript levels of PsHSF1 in vegetative hyphae treated with 0.5 mM t-BHP for 5, 15 and 60 min (B1, B2 and B3, respectively) relative to untreated hyphae (CK). B. Expressions level of PsHSF1 in vegetative hyphae incubated at 30°C for 15, 30 and 90 min (T1, T2 and T3, respectively) relative to the level in hyphae kept at 25°C (CK). C. Expression levels of PsHSF1 in different life stages (MY: mycelium, SP: sporangia, ZO: zoospore, CY: cysts, GC: germinated cysts) and during growth in planta 1.5, 3, 6, 12 and 24 h after inoculation (IF1.5–IF24). Mycelium was inoculated on leaves of soybean cultivar Williams. The transcript level in MY was set at 1. Phytophthora sojae ActinA gene was used as endogenous gene. Real-time PCR was replicated biologically three times.

reaction (SqRT-PCR) was also applied and led to a similar result (Fig. S4A). Also heat stress (exposure to 30°C; Fig. 2B) induced expression of PsHSF1, but in contrast to the transient pattern seen with oxidative stress, heat

To further investigate the function of PsHSF1, we generated PsHSF1-silenced transformants by polyethylene glycol (PEG)-mediated co-transformation (Hua et al., 2008). One hundred forty transformants were recovered that were analysed for the presence of the construct. Subsequently, qRT-PCR was used to determine PsHSF1 transcript levels in germinating cysts. Three transformants had strongly reduced PsHSF1 RNA levels (> 90%) (Fig. S5), two of which, T27 and T58, are shown here in the results. T15, the third one, behaved overall similar as T27 and T58. They all showed no reduction in mycelial growth on Plich medium for 7 days at 25°C when compared with the wild-type strain (WT) and a control transformant (Green Fluorescent Protein (GFP)-expressing transformant; CK) (Fig. 3A and B). In contrast, treatment with 2 mM H2O2 caused a 60% reduction of mycelial growth in the PsHSF1-silenced transformants, when compared with WT and CK (Fig. 3A and B). T27 and T58 were highly sensitive to H2O2 and not able to grow in the presence of 5 mM H2O2, while WT and CK could still proliferate (Fig. 3A and B). Moreover, the PsHSF1-silenced transformants showed a reduced growth rate when incubated at 30°C (Fig. 3A and B). Taken together, these results indicate that PsHSF1 is essential for proper growth of P. sojae under oxidative and heat stress. PsHSF1 has a role in cyst germination and virulence We next studied the behaviour of the P. sojae PsHSF1silenced transformants throughout the life cycle. The transformants T27 and T58 had similar growth rates and colony morphology as WT and CK when cultured on 10% V8 plates at 25°C. No significant differences were observed for zoospore production, encystment and oospore production (Table S3). This shows that vegetative development and sexual development is not dependent on PsHSF1. In contrast, cyst germination was affected in PsHSF1-silenced transformants. Already after 1.5 h incubation, the difference was significant with ∼ 50% of WT



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Fig. 3. PsHSF1-silenced transformants are hypersensitive to H2O2 and high temperature. A. Mycelial growth of the wild type strain P6497 (WT), control strain (CK) and the PsHSF1-silenced transformants T27 and T58 on Plich medium without H2O2 or supplemented with 2 or 5 mM H2O2 and cultured at 25°C or Plich medium without H2O2 cultured at 30°C. B. The mycelia growth rate in mm day−1 (Y-axis) was measured and subjected to statistical analysis. Three repeats were performed with similar results. Error bars represent the standard deviations and asterisks represent significant differences (Student’s t-test, P < 0.01).

and CK cysts germinating and only 20% of the cysts harvested from the PsHSF1-silenced transformants, and after 6 h incubation, the difference was still significant (∼ 80% versus ∼ 40%) (Fig. 4A and B). This mutant phenotype is consistent with the high transcript level at the cyst germination stage (Fig. 2C). To determine the role of PsHSF1 in pathogenicity, we performed disease assays. Zoospores of WT, CK and the PsHSF1-silenced transformants T27 and T58 were inoculated on etiolated seedlings of the susceptible soybean cultivar Williams at 25°C. To compensate for the reduced cyst germination rate, we doubled the amount of zoospores of T27 and T58 in the inoculums. T27 and T58 produced necrotic dark brown spots at 36 h after inoculation, whereas the WT and CK strains induced spreading lesions (Fig. 4C). Biomass quantification confirmed that

silencing of PsHSF1 hampers the ability of P. sojae to successfully colonize the host. The amount of P. sojae DNA in soybean hypocotyls inoculated with T27 and T58 was reduced over 50-fold when compared with WT and CK (Fig. 4D). These results suggest that PsHSF1 is required for virulence. PsHSF1 is required for detoxifying the plant oxidative burst and suppressing callose deposition To gain further insight into the role of PsHSF1 in the infection process, we analysed the infected seedlings in more detail at the microscopic level. Based on the necrotic brown spots and cell death observed on the T27 and T58 inoculated hypocotyls (Fig. 4C), we hypothesized that PsHSF1-silenced transformants are incapable of


1356 Y. Sheng et al. Fig. 4. PsHSF1-silenced transformants showed reduced cyst germination rate and reduced virulence. A. Cyst germination of wild type strain P6497 (WT), control strain (CK) and PsHSF1-silenced transformants T27 and T58. Images were captured after 3 h incubation on 0.5% V8 medium at 25°C. Bar = 50 μm. B. Cyst germination rates after 1.5, 3, 4.5 and 6 h incubation were calculated based on counting 100 cysts, and subjected to statistical analysis based on two-tailed t-test (P < 0.01). Three repeats were performed with similar results. The Y-axis shows the ratio, the error bars represent the standard deviations and the asterisks the significant differences. C. Etiolated seedlings of susceptible soybean cultivar Williams inoculated with zoospores from WT, CK, T27 and T58 and photographed at 36 h after inoculation. The experiments were carried out three times with the similar result. D. Relative virulence expressed as the ratio between the amount of P. sojae DNA and soybean DNA detected 36 h after inoculation of etiolated seedlings. Values marked with an asterisk are significantly different with the wild type strain (WT) which was set at 1, and the control strain (CK) (P < 0.01).

circumventing plant defence responses. Accordingly, we monitored defence responses by analysing callose deposition using aniline blue staining. Very low or undetectable levels of callose deposition were observed after 42 h in soybean epidermal cells when inoculated with the WT strain. In contrast, in T58 inoculated seedlings, the epidermal cells showed a strong fluorescence signal pointing to the accumulation of phenolic compounds and callose deposition (Fig. 5A). Since PsHSF1 is upregulated during oxidative stress and ROS are known to play a role in many plant– pathogen interactions, ROS accumulation was monitored around the infection sites. In soybean epidermal cells inoculated with WT (Fig. S1) and T58 zoospores, accumulation of H2O2 was observed in the early stages of the interaction. At 3 and 6 h post-infection, no obvious differences were observed between tissues inoculated with the WT and T58 (data not shown). The progress of colonization and H2O2 accumulation was comparable indicating that the ability to penetrate and colonize and to trigger plant defence responses in the early infection

phase is not disturbed when PsHSF1 is absent. However, later during the interaction, the difference between WT and T58 became evident. At 42 h, the massive infection caused by the WT strain was not hampered by ROS. No H2O2 accumulation was detectable suggesting that the wild type strain can scavenge the ROS produced by the host to establish a successful infection. In contrast, in soybean epidermal cells inoculated with T58, H2O2 was still present in the later stages of the interaction (Fig. 5B) suggesting that the PsHSF1silenced transformant is incapable of scavenging hostderived ROS and to suppress basal resistance of the host. To validate that silencing of PsHSF1 compromises ROS scavenging, we supplemented T58 with an artificial ROS scavenger. At 6 h after inoculation with T58 zoospores, we added exogenous GSH at the inoculation site and analysed the H2O2 accumulation 36 h later. GSH treatment resulted in strongly reduced H2O2 levels and was accompanied by an increase in growth of invasive hyphae (Fig. 5B). The finding that virulence of T58 is restored by



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Fig. 5. PsHSF1-silenced transformants are unable to suppress callose deposition and production of ROS. A. Callose deposition detected by aniline blue staining in soybean hypocotyls inoculated with the zoospores of the P. sojae wild type strain P6497 (WT) and PsHSF1-silenced transformant T58 at 42 h after inoculation. The epidermis of stained hypocotyl was examined with an Olympus 1X 71 inverted microscope. The blue fluorescence was detected using UV filter (Excitation at 330–385 nm, emission at 420 nm) and differential interference contrast (DIC) microscopy. Bar = 20 μm. B. ROS accumulation in infected hypocotyls 42 h after inoculation with or without GSH treatment was detected by DAB staining. GSH was applied at the site of inoculation 6 h after inoculation. The epidermis of stained hypocotyls was examined with an Olympus 1X 71 inverted microscope. Bar = 20 μm.

an exogenously added ROS scavenging compound implies that PsHSF1 plays a prominent role in detoxifying the plant oxidative burst.

Silencing of PsHSF1 attenuates the activity of extracellular peroxidases and laccases Since we speculated that PsHSF1 regulates the expression of peroxidase and laccase genes, we compared the extracellular peroxidase and laccase activity of control strains and PsHSF1-silenced transformants. For peroxidase activity, we analysed the degradation of Congo Red (CR) and, as shown in Fig. 6A, reduced degradation halos were observed for T27 and T58 when grown on Plich medium supplemented with 500 μg ml−1 CR. For laccase activity, we added ABTS [2,2′-azino-bis (3-ethylbenzothiazoline-6)-sulphonic acid] as substrate to solid lima bean agar (LBA) medium. In comparison to the control strains, T27 and T58 accumulated reduced amounts of oxidized ABTS resulting in less dark purple staining underneath the mycelial mat (Fig. 6C). These results show that indeed, the extracellular peroxidase and laccase activity are reduced in the PsHSF1-silenced transformants. To explore whether this reduction in enzyme activity was due to deregulation at the transcriptional level, we examined the transcript levels of several peroxidase and laccase genes. We selected the genes based on annotation in the P. sojae genome and their expression profiles in WT deduced from RNA-seq data (Ye et al., 2011). This resulted in nine peroxidase genes, most of which are upregulated during infection (Fig. S6B). All nine are predicted to encode a protein possessing a signal peptide by the SignalP program. Phytophthora sojae

contains 15 genes that are predicted to encode a laccase with a Cu-oxidase domain (IPR001117, IPR011706 and IPR011707) (Fig. S6A). Five of these, all with a signal peptide (PsLAC1: Ps129757; PsLAC2: Ps129758; PsLAC3: Ps129759; PsLAC4: Ps137983; PsLAC5: Ps137984), show differential expression during the life cycle while the others show low levels of expression during all stages (Fig. S6A). To compare expression in WT and T58 of the nine peroxidase genes and the five differentially expressed laccase genes during oxidative stress (5 mM H2O2), we performed qRT-PCR. In T58, the transcript levels of three peroxidase genes (Ps138408, Ps139488 and Ps139538) (Fig. 6B) and two laccase genes (PsLAC4 and PsLAC5) were significantly decreased (Fig. 6D) (30–56% and 83–92% respectively). Since silencing of PsHSF1 affects transcript levels, we infer that PsHSF1 acts as a TF that directly or indirectly regulates the transcription of these peroxidase genes and laccase genes during oxidative stress.

Silencing of the laccase gene PsLAC4 attenuates pathogenicity and laccase activity To elucidate whether laccase activity contributes to P. sojae virulence, we transiently silenced PsLAC4, a laccase gene of which the transcription is strongly reduced in the PsHSF1-silenced transformant (Fig. 6D). Twenty PsLAC4 dsRNA-treated lines were analysed for laccase activity. These assays were carried out at day 9–14 after transformation; zoospores for pathogenicity assays were collected at day 11 after transformation and mycelium for RNA isolation at day 13. qRT-PCR revealed four transformants which showed a relatively high silencing efficiency with a reduction in PsLAC4 transcript levels


1358 Y. Sheng et al. Fig. 6. In PsHSF1-silenced transformants the peroxidase and laccase activity is reduced. A. Peroxidase activity of the wild type strain P6497 (WT), control strain (CK) and the PsHSF1-silenced transformants T27 and T58 determined by discoloration of Congo Red (500 μg ml−1) in Plich medium after 24 h incubation. B. Relative expression of nine peroxidase-encoding genes in WT (set at 1) and transformant T58 under oxidative stress conditions (5 mM H2O2, for 5 min). C. Laccase activity determined by monitoring oxidized ABTS (purple color) in LBA media supplemented with 0.2 mM ABTS after 7 days of incubation. D. Relative expression of five laccase-encoding genes in WT (set at 1) and transformant T58 under oxidative stress conditions (5 mM H2O2, for 5 min).

of 23–58% (Fig. 7C). These four not only showed reduced laccase activity, as demonstrated by the reduced dark purple staining in medium containing ABTS (Fig. 7B), but they also showed attenuated pathogenicity. Zoospore inoculation on etiolated seedlings of susceptible soybean

cultivar Williams resulted in necrotic dark brown lesions and reduced lesion sizes 48 h after infection when compared with WT and CK control strains (Fig. 7A and D). These results show that PsLAC4 is required for full pathogenicity. In case PsHSF1 indeed regulates expression of

Fig. 7. Transient silencing of PsLAC4 results in reduced laccase activity and attenuated pathogenicity. A. Zoospores of wild type strain P6497 (WT), control strain (CK) and four PsLAC4 transformants subjected to transient transformation with a dsRNA targeting PsLAC4, were inoculated on etiolated seedlings of the susceptible soybean Williams. The zoospores were harvested and inoculated 11 days after the transient transformation. Two days later photographs were taken. B. Laccase activity of the same strains was assayed by monitoring oxidized ABTS (purple color) after growth for 7 days on LBA medium containing 0.2 mM ABTS. Mycelium was transferred to ABTS medium 9 to 14 days after transient transformation. C. The efficiency of silencing was determined by qRT-PCR in mycelium harvested 13 day after transient transformation. D. Lesion length of soybean seedlings inoculated with WT, CK and four PsLAC4 transformants for two days.


PsHSF1, a ROS response regulated in Phytophthora sojae PsLAC4, then the reduction of PsLAC4 activity may be one of the reasons that PsHSF1-silenced transformants show reduced pathogenicity. To know how PsHSF1 potentially regulates the expression of PsLAC4, we searched for characteristic heat shock elements in the promoter region of PsLAC4. At 135–105 bp upstream of the translation initiation start codon of PsLAC4, there are four ‘nTTCn’ repeats (TTCnnnnnnTTCnnnnnTTCnnnnnTTCA). Similarly, at 126–109 bp upstream of the translation initiation start codon of PsLAC5, three ‘nTCCn’ repeats were detected (TTCnnnnTTCnnTTC). These repeats are absent in the other laccase genes. Besides, these elements are also present in the promoters of the PsLAC4 and PsLAC5 homologues in P. ramorum and P. infestans (Fig. S7). Based on these findings, we postulate that PsHSF1 regulates PsLAC4 and PsLAC5 expression by binding to these elements. Discussion TFs play a key role in the regulation of gene expression in all organisms. In plant pathogenic fungi, ROS tolerancerelated TFs induce or repress gene expression in order to counteract the oxidative burst that takes place at the site of invasion during the initial stage of the interaction between fungus and host plant (Lin et al., 2009; Guo et al., 2010; 2011). In this study, we demonstrate that, similar to plant pathogenic fungi, the oomycete P. sojae can overcome this plant defence response by the production of ROS-detoxifying enzymes. In Saccharomyces cerevisiae, at least five TFs are involved in the response to oxidative stress (HSF1, YAP1, SKN7 and MSN2/4) (Lee et al., 1999; Raitt et al., 2000; Hasan et al., 2002). Homologues of these TFs are also denoted to play distinctive roles in oxidative stress tolerance in other fungi (Cuéllar-Cruz et al., 2008; Guo et al., 2011). The ubiquitous presence of ROS-responsive TFs emphasizes the indisputable status of these proteins in response to redox challenges. Although the genome-wide inventory of ROS-responsive TFs in Phytophthora species failed to identify homologues of YAP1, SKN7 and MSN2/4, a large family of HSF genes was identified ranging from 18 members in P. ramorum, 22 in P. sojae and up to 24 in P. infestans. This contrasts with the number in yeast and Drosophila, with only a single HSF gene (Sorger and Pelham, 1988; Jedlicka et al., 1997). Yeast HSF1 not only plays roles in stress resistance, but is also required for cell growth and differentiation (Morano and Thiele, 1999). Vertebrates contain four HSF genes that are involved in different biological processes, including stress tolerance, cell differentiation and development (Morimoto, 1998), and plants have even more, e.g. tomato has 18 HSF genes and Arabidopsis has 21 HSF

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genes (Nover et al., 1996; 2001). The Phytophthora HSF proteins have only limited homology to plant, fungal and animals HSFs. The large number of HSF genes in Phytophthora seems to be the result of ancient duplication events; they share significant homology with several pairs located on the same scaffold close to each other and a group of six clustered on the genome. This clustering is reminiscent of the genome organization of other gene families in Phytophthora such as the phospholipase D family and the nuclear LIM interactor-interacting factors (NIFs) family (Tani et al., 2005; Meijer et al., 2011). The Phytophthora HSF-regulated network may function with high redundancy and a certain degree of complexity to respond to environmental stress conditions or act during specific life stages. However, the significance of gene clustering in this process remains unknown. The transcripts of PsHSF1 strongly and rapidly accumulated under oxidative stress conditions, and consistently, PsHSF1-silenced transformants displayed more sensitivity to H2O2 than the control strains. This result clearly pointed to a role for PsHSF1 in oxidative stress, strengthening the idea that the attenuated pathogenicity of the PsHSF1-silenced transformants is caused by the fact that they cannot handle the ROS that accumulate around the infection site. The finding that application of exogenous GSH, a H2O2 reducing agent, can compensate for the absence of PsHSF1 in expanding lesions demonstrates that the ability of P. sojae to scavenge host plantderived ROS is controlled by PsHSF1. Thus, it is likely that the attenuated pathogenicity of the PsHSF1-silenced transformants is due to the strong plant defence response and that PsHSF1 is essential for detoxifying the plant oxidative burst. ROS, produced by plasma membranelocalized NADPH oxidases as by-products of various metabolic pathways (Doke et al., 1996), are scavenged by antioxidative defence components. The ROS levels are kept in equilibrium by production and scavenging under physiological steady state conditions but are perturbed during pathogen invasion. The oxidative burst, the rapid release of ROS species, is one of the earliest defence responses at the invasion site when attacked by pathogen (Apostol et al., 1989). It was first reported by demonstrating that a plasma membrane located novel O2−generating NADPH oxidase system in potato was activated in an incompatible interaction with P. infestans whereas a compatible interaction failed to induce O2− accumulation (Doke, 1985). Subsequently, several studies reported ROS generation during incompatible plant–pathogen interactions (Bestwick et al., 1997), and this is now regarded as a hallmark of successful pathogen recognition by the plant (Nürnberger et al., 2004). In this study, however, we also observed ROS accumulation in a compatible interaction. During the early phase of infection of soybean by P. sojae, ROS was produced but in the next


1360 Y. Sheng et al. phase, production ceased resulting in the absence of ROS during the late infection phase. Thus, both compatible and incompatible interactions can trigger a rapid plant defence response resulting in an oxidative burst. Virulent wild-type P. sojae strains sense the initial production of ROS by the host plant and initiate counteractive mechanisms. As a result, ROS are detoxified and the plant defence response is suppressed. ROS sensing and initiation of counteractive measures is, at least in part, dependent on PsHSF1 since PsHSF1-silenced transformants failed to reduce the ROS accumulation at the later stage of invasion. Although the ROS scavenging ability was impaired, the pathogenicity of the strain was not affected since exogenous application of the reductant GSH restored virulence. This also emphasizes that the soybean defence response against P. sojae is highly dependent on the oxidative burst. PsHSF1 seems to be essential for regulating the ROS detoxification and detoxifying the plant oxidative burst. In most plant pathogens, secreted peroxidases are important for plant-derived ROS scavenging during plant–pathogen interactions (Molina and Kahmann, 2007; Howlett et al., 2009). Since PsHSF1-silenced transformants became more sensitive to oxidative stress and ROS accumulation was observed at late infection stages, we assumed that the ROS scavenging ability was compromised which was confirmed by a reduction in peroxidase and laccase activity. In agreement with this, the transcript levels of several genes encoding extracellular peroxidase and laccases were significantly reduced in a PsHSF1-silenced transformant. Laccase has been identified as an important virulence factor in several fungi (Bar Nun et al., 1988; Mayer and Staples, 2002). In this study, the contribution of laccase activity to P. sojae virulence was shown by transient silencing of PsLAC4. Silencing resulted in reduced laccase activity levels and decreased pathogenicity although less severe as detected for PsHSF1-silenced transformants. This suggests that the loss of laccase activity is a major component of the PsHSF1 silencing phenotype but that other enzymatic activities regulated by PsHSF1 also contribute. Most likely, the peroxidases that were affected in their expression by PsHSF1 silencing are among these. The promoter regions of laccase genes have several putative cis-acting elements such as xenobiotic-responsive elements, metal-responsive elements and stress-responsive elements (Xiao et al., 2006). Although HSF proteins display a wide variability between different species, the heat shock elements in promoters that are targeted by HSF trimers are well conserved from yeast to humans. They consist of three contiguous repeats of the short sequence 5′-nGAAn-3′ (Xiao and Lis, 1988; Xiao et al., 1991; Wu, 1995). Multiple inverted repeats of nGAAn (nTTCn) were found at the promoter region of the two

most downregulated laccase genes PsLAC4 and PsLAC5, as well as their homologues in P. ramorum and P. infestans. Based on these findings, we postulate that PsLAC4 and PsLAC5 might be the target genes that are directly regulated by PsHSF1 upon binding of these promoter elements. Taken together, we have identified the P. sojae TF PsHSF1 as a major player in oxidative stress and ROS depletion during infection of soybean. Furthermore, we identified some potential downstream target genes of PsHSF1. This research contributes to a better understanding of transcriptional regulation in oomycetes. Experimental procedures Phytophthora sojae growth conditions and tissue harvest Phytophthora sojae strain P6497 (race 2) provided by Professor Brett Tyler (OSU’s Center for Genome Research and Biocomputing, OR, USA) and all transgenic lines in this study were routinely grown on 10% V8 media at 25°C in the dark (Erwin and Ribeiro, 1996). The asexual life stages such as vegetative hyphae, sporulating hyphae, zoospores, cysts and germinated cysts were collected as described (Ye et al., 2011). Tissues of infections stages (P. sojae–soybean interaction) were prepared by inoculating 107 zoospores on the hypocotyls of etiolated soybean seedlings of cultivar Williams at 25°C, and the infected plant tissue were harvested at 1.5 h, 3 h, 6, 12 h and 24 h respectively. All tissues were immediately frozen in liquid N2 followed by grinding for generating RNA samples for expression analysis using qRT-PCR as described (Ye et al., 2011). Hyphae cultured in 10% V8 juice at 25°C in the dark for 3 days were washed with Plich liquid medium (van West et al., 1999) followed by incubation in 20 ml of Plich liquid medium. After 0, 45 or 55 min, H2O2 or t-BHP was added to the Plich liquid medium to a final concentration of 5 mM or 0.5 mM, respectively, and at 60 min, all samples were harvested at the same time. In this way, all samples were kept equally long in Plich liquid medium before they were frozen in liquid N2 and grinded for RNA extraction.

Phytophthora sojae transformation The full length of PsHSF1 open reading frame was amplified with PrimeStar polymerase (TaKaRa) from genomic DNA (gDNA) of P6497 and ligated in antisense orientation into the pHAM34 vector. Phytophthora sojae transformation was carried out as described (Hua et al., 2008). The P. sojae transformants were screened for PsHSF1 transgene by amplifying gDNA of each line as described (Hua et al., 2008). Total RNA of the transformants was extracted from germinated cysts and qRT-PCR assay was performed to analyse the transcript levels. Double-stranded RNA of PsLAC4 was synthesized according to the protocol of the Ambion MEGAscript RNAi kit. Transient transformation was carried out using the PEG-mediated method but with some modifications as described (Zhao


PsHSF1, a ROS response regulated in Phytophthora sojae et al., 2011). All phenotypic assays were carried out at day 8–15 after transformation. Total RNA of selected transformants mycelium was extracted on day 11 and day 13 and qRT-PCR assay was performed to test the silencing efficiency.

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in ethanol (96%) for 48 h (Molina and Kahmann, 2007). To examine the effect of treatment with an exogenously added reductant on H2O2 accumulation, 0.25 mM GSH was added at the site of inoculation 6 h after inoculation with zoospores; DAB staining was performed 36 h later. The epidermis of the treated hypocotyls was examined using an Olympus 1X 71 inverted microscope.

Oxidative and heat shock sensitivity assay The sensitivity of PsHSF1-silenced transformants to hydrogen peroxide and high temperature (30°C) was evaluated on modified Plich medium (van West et al., 1999) plates. After 4 days of growth on 10% V8 at 25°C, 5 mm hyphal plugs from the wild-type strain WT, the GFP-expressing transformant CK and PsHSF1-silenced transformants were transferred to modified Plich medium amended without or with 2 mM or 5 mM H2O2 and incubated at 25°C for 7 days. Plates without H2O2 were incubated at 25°C or 30°C for 7 days. The experiment was repeated three times with each assay in triplicate.

Extracellular enzyme activity assay Laccase activity on solid medium was measured according to Guo and colleagues (2011) with some modifications. 5 × 5 mm hyphal tip plugs were inoculated on LBA medium supplemented with 0.2 mM 2, 29-azino-di-3ethylbenzathiazoline-6-sulfonate (ABTS, Sigma-Aldrich, USA) and incubated for 7 days at 25°C in the dark. To detect peroxidase secretion, 5 × 5 mm hyphal tip plugs were placed on Plich medium containing 500 μg ml−1 CR (Sigma-Aldrich, USA) for 24 h at 25°C in the dark. The experiments were repeated three times, and within each experiment, there were three replicates for each strain.

Cyst germination assay Zoospores were harvested as described (Ye et al., 2013) and transferred to tubes. The tubes containing at least 20 μl−1 zoospores in a 500 μl suspension were vortexed for 90 s to induce encystment. Five microlitre droplets of the cyst suspension were transferred to 1% agar plate with 0.5% V8 and incubated at 25°C for 1.5, 3, 4.5 and 6 h respectively to allow germination. The germination of at least 100 cysts was examined for each line, and all assays were replicated three times with each assay in triple.

Virulence assay The virulence was tested by zoospore inoculation of the hypocotyls of etiolated soybean seedlings of cultivar Williams, a cultivar compatible with P. sojae strain P6497 (Li et al., 2013). The inoculated soybean seedlings were maintained in 80% humidity and darkness at 25°C for 48 h before photographs were taken. The virulence was quantified by determining the ratio of P. sojae DNA to soybean DNA in the infected tissues, measured by qRT-PCR (Wang et al., 2011).

Aniline blue and diaminobenzidine (DAB) staining Aniline blue staining was performed to visualize the accumulation of phenolic compounds in inoculated soybean hypocotyls. The hypocotyls were stained 42 h after inoculation with zoospores in an aniline blue solution of 0.005% for 30 min. Epidermal cells were examined using an Olympus 1X71 inverted microscope. Blue fluorescence was detected using UV filter (excitation at 330–385 nm, emission at 420 nm) and differential interference contrast (DIC) microscope. To visualize the accumulation of hydrogen peroxide (H2O2) in the infected plant tissue, DAB staining was performed. Briefly, soybean hypocotyls were collected 42 h after inoculation and were placed in a 1 mg ml−1 solution of DAB for 16 h in the dark at room temperature followed by decolourization

Nucleic acid manipulation and RT-PCR assay gDNA of P. sojae strain P6497 and the putative transformants strains was isolated from hyphae grown in 10% V8 liquid medium using a plant DNA kit (OMEGA). To quantify the virulence of WT and silenced transformants, the ratio of P. sojae DNA to soybean DNA in the infected tissues was measured by qRT-PCR. Total gDNA of tissue sampled from five seedlings was extracted using a DNeasy plant mini kit (Qiagen) following the recommended protocol. Phytophthora sojae ActinA gene [Joint Genome Institute (JGI) GeneID: 108986] was used as the pathogen target and soybean housekeeping gene CYP2 (TC224926) was used as the host endogenous gene (Wang et al., 2011). Total RNA of P. sojae from distinct stages of the life cycle, from infection stages and from hyphae exposed to different treatments, was isolated by the NucleoSpin RNA II extraction kit (Macherey-Nagel) following the procedures described by the manufacturer. The integrity of total RNA was confirmed using agarose gel electrophoresis. The RNA was quantified using a spectrophotometer (Nanodrop ND-1000). To investigate the transcript levels of target genes, SqRTPCR (Hua et al., 2008) and qRT-PCR were carried out. First-strand cDNA was synthesized using M-MLV reverse transcriptase (RNase-free, Invitrogen) and oligo dT 18 primer (Invitrogen). Reactions of qRT-PCR were performed on an ABI PRISM 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) 95°C for 30 s, 40 cycles of 95°C for 5 s, 60°C for 34 s to calculate cycle threshold (Ct) values, followed by 95°C for 15 s, 60°C for 1 min, then 95°C for 15 s to obtain melt curves. The 7500 System Sequence Detection Software was used to obtain relative expression levels of each sample. Primers used in this study were designed by Primer 3.0 Input (version 0.4.0) and commercially synthesized (Invitrogen, Shanghai, China). The primer pairs are listed in Table S2. The ActinA gene from P. sojae was used as a constitutively expressed endogenous control and ΔΔCt method was used to calculate the relative


1362 Y. Sheng et al. transcription level. All experiments were performed at least three times with independent RNA isolations.

Bioinformatics analysis Phytophthora sojae and P. ramorum DNA and protein sequence predictions were obtained from the JGI (http:// www.jgi.doe.gov/); P. infestans DNA and protein sequences were retrieved from the Broad Institute (http://www.broad .mit.edu/). BLAST searches were performed against these genome sequence databases (Altschul et al., 1990). Detailed information on Phytophthora HSF genes is summarized in Table S1. Protein sequences of S. cerevisiae HSF1 (CAA96777) and Arabidopsis thaliana HSF1 (NP_193510) were used in TBLASTN and PSI-BLAST searches of the above-mentioned sequenced genomes (McGinnis and Madden, 2004). Other sequences of HSFs used for phylogenetic analysis were obtained from the protein databases at NCBI (http://www.ncbi.nlm.nih.gov/). The candidate gene models were submitted to Prosite pattern available on the web (http://www.expasy.org/prosite), Pfam (http://pfam.sanger.ac.uk/) and NCBI-CDD (http://www.ncbi .nlm.nih.gov/Structure/cdd) to discriminate conserved functional sites. NES and NLS were analysed by the NetNES1.1 Server (La Cour et al., 2003) and NLStradamus (Ba et al., 2009). Sequence alignments were made with the Clustal_W program (Thompson et al., 1994) and the phylogenetic dendrograms were constructed by the MEGA 5.0 program (Tamura et al., 2007) with neighbour-joining algorithms using 1000 bootstrap replications.

Acknowledgements This work was supported in part by grants from China National Funds for Distinguished Young Scientists (31225022), “111” project from Ministry of Education of China and Special Fund for Agro-Scientific Research in the Public Interest (201303018) of China to Yuanchao Wang, and by grants from the Netherlands Organization for Scientific Research (NWO) in the framework the China-Netherlands Joint Research Project of Life Sciences department (ALW) (833.13.002/ 2013DFG32030) to Francine Govers and Yuanchao Wang and a project VIDI (Visualising the Impact of the legislation by analysing public DIscussions using statistical means) (Technology Foundation STW 10281) to Harold J. G. Meijer.

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Supporting information Additional Supporting Information may be found in the online version of this article at the publisher’s web-site: Fig. S1. ROS accumulation in soybean during early stages of infection. Fig. S2. PsHSF1 (Ps142192) is located in a cluster of HSF genes. Fig. S3. Transcriptional analysis of P. sojae PsHSF genes during oxidative stress. Fig. S4. Transcript levels of PsHSF1 under oxidative stress and during asexual life stages. Fig. S5. Silencing efficiency in PsHSF1-silenced transformants. Fig. S6. Expression profiles of P. sojae putative laccaseencoding genes and peroxidase-encoding genes. Fig. S7. Potential heat shock elements in the promoter regions of Phytophthora LAC4 and LAC5 genes. Table S1. Overview of HSF genes in P. sojae, P. infestans and P. ramorum. Table S2. Primers for qRT-PCR assays and cloning. Table S3. Characteristics of PsHSF1-silenced mutants T27 and T58 in comparison to control strains (WT and CK).


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