Overexpression of a Phytophthora Cytoplasmic CRN Effector Confers Resistance to Disease, Salinity and Drought in Nicotiana benthamiana

Abstract

The Crinkler (CRN) effector family is produced by oomycete pathogens and may manipulate host physiological and biochemical events inside host cells. Here, PsCRN161 was identified from Phytophthora sojae based on its broad and strong cell death suppression activities. The effector protein contains two predicted nuclear localization signals and localized to nuclei of plant cells, indicating that it may target plant nuclei to modify host cell physiology and function. The chimeric gene GFP:PsCRN161 driven by the Cauliflower mosaic virus (CaMV) 35S promoter was introduced into Nicotiana benthamiana. The four independent PsCRN161-transgenic lines exhibited increased resistance to two oomycete pathogens (P. parasitica and P. capsici) and showed enhanced tolerance to salinity and drought stresses. Digital gene expression profiling analysis showed that defense-related genes, including ABC transporters, Cyt P450 and receptor-like kinases (RLKs), were significantly up-regulated in PsCRN161-transgenic plants compared with GFP (green fluorescent protein) lines, implying that PsCRN161 expression may protect plants from biotic and abiotic stresses by up-regulation of many defense-related genes. The results reveal previously unknown functions of the oomycete effectors, suggesting that the pathogen effectors could be directly used as functional genes for plant molecular breeding for enhancement of tolerance to biotic and abiotic stresses.

Introduction

The dual roles of effectors secreted by pathogens have been extensively reported (Kamoun 2006). Effectors generally facilitate infection by suppressing plant immune responses or modifying host physiology, resulting in effector-triggered susceptibility (ETS). However, effectors can also be recognized as avirulence proteins and activate plant immune responses, known as effector-triggered immunity (ETI), in the presence of the corresponding immunoreceptors (Kamoun 2006, Hogenhout et al. 2009). ETI often results in a hypersensitive response (HR) that prevents the spread of pathogen infection (Thomma et al. 2011).

Phytophthora species belong to a phylogenetically distinct group of eukaryotic microorganisms classified as oomycetes, and cause devastating diseases on numerous crops and ornamental plants. The most notorious species is P. infestans that was responsible for the Irish potato famine in the 1840s and is still a serious threat to sustainable potato and tomato production worldwide (Fry 2008). Another species, P. sojae, which causes substantial yield losses annually is one of the predominant pathogens of soybean (Glycine max) (Tyler 2001). These pathogens are usually hemibiotrophic with an initial biotrophic phase during early infection followed by a necrotrophic phase in late infection stages. This process is probably achieved by sequential delivery of effectors with distinct activities into host cells (Koeck et al. 2011, Wang et al. 2011, Kleemann et al. 2012).

A large number of effectors have been identified in oomycete pathogens. Among them, two classes of cytoplasmic effectors [RxLR and Crinkler (CRN)] were well characterized. RxLR effectors contain the conserved motif RxLR (arginine, any amino acid, leucine, arginine) in their N-termini, which delivers effectors into host cells (Whisson et al. 2007, Dou et al. 2008a). Nearly all avirulence genes cloned from oomycete pathogens encode RxLR effectors (Jiang and Tyler 2012). The discovery of the RxLR motif in oomycetes has accelerated the cloning process of avirulence genes (Vleeshouwers et al. 2008, Dong et al. 2009). Although the RxLR effectors were initially identified based on their avirulence activity on plant cultivars containing cognate R genes, it has also been reported that these effectors contribute to virulence in susceptible backgrounds (Dou et al. 2008b, Bos et al. 2010, Dong et al. 2011). CRNs that were firstly reported as crinkling and necrosis-inducing proteins (Torto et al. 2003) also contribute to virulence by modulating distinct physiological events in host cells (Liu et al. 2011, Shen et al. 2013, Stam et al. 2013, Mafurah et al. 2015). Similar to the RxLR effector, the N-terminus of CRN contains a conserved FLAK (phenylalanine, leucine, alanine, lysine) motif that translocates effector protein inside plant cells (Schornack et al. 2010). However, CRN effectors have some unique features that are not found in RxLR effectors. First, CRN effectors are highly conserved in oomycete pathogens and are widely distributed in all oomycetes (Schornack et al. 2010) and even in the chytrid fungus Batrachochytrium dendrobatidis (Joneson et al. 2011), suggestive of basic roles for CRNs in oomycete parasitism. Unlike CRNs, RxLR effectors were only found in members of the Peronosporales lineage (Tyler et al. 2006, Levesque et al. 2010, Links et al. 2011. Secondly, the expression levels of CRNs are much higher during infections than those of RxLR effectors (Haas et al. 2009, Shen et al. 2013). Thirdly, the majority of CRNs are localized to the plant nucleus (Schornack et al. 2010, Stam et al. 2013), suggestive of a role in interfering with plant nuclear signaling during infection.

Several virulence-related CRNs have been identified and characterized. Both P. infestans CRN8 and P. sojae CRN63 can trigger plant cell death and promote plant susceptibility. Interestingly, CRN8 exhibits kinase activity, which is not required for cell death induction but is essential for virulent function (van Damme et al. 2012). Similar to the Cucumber mosaic virus (CMV) 2b protein, CRN63 perturbs plant reactive oxygen species (ROS) homeostasis through directly interacting and interfering with plant catalases (Zhang et al. 2015a). Expression of many other CRNs rarely leads to cell death in plants, but may inhibit cell death triggered by elicitors (Shen et al. 2013, Stam et al. 2013). For example, P. sojae CRN70 suppresses plant cell death, H₂O₂ accumulation and expression of defense-associated genes (Rajput et al. 2014). Phytophthora sojae CRN115, sharing high sequence similarity with PsCRN63, also interacts with plant catalases, but protects plant cells from death by enhanced ROS scavenging (Zhang et al. 2015a). Recently, we showed that PsCRN115 confers disease resistance and tolerance to abiotic stresses when it is expressed in plants (Zhang et al. 2015b).

Plants are constantly challenged by various biotic and abiotic stresses throughout their life cycle. These stresses adversely affected plant growth and reproduction, resulting in substantial yield losses annually (Vij and Tyagi 2007, Helliwell et al. 2013). Substantial efforts have been made to confer resistance to pathogens and improve tolerance to abiotic stress, among which an important approach is to generate genetically engineered plants with increased resistance. Many pathogen components, such as Avr genes and bacterial Hrp proteins, have been reported as candidate genes to improve disease resistance. Some of these genes can be expressed under control of a pathogen-inducible promoter that can limit gene expression in desirable regions and time where/when a virulent pathogen is present (McDowell and Woffenden 2003, Gurr and Rushton 2005). The bacterial Hrp proteins not only elicit plant resistant responses but also promote growth of plant aerial parts (Fu et al. 2014). Several Hrp proteins have been successfully used to induce disease resistance in several crops, including rice, wheat and Chinese cabbage (Chen et al. 2008, Sun et al. 2010, Fu et al. 2014). Expression of elicitins from oomycete pathogens enhances plant resistance to disease and abiotic stress (Keller et al. 1999, Jiang et al. 2004).

Here we identified a CRN effector PsCRN161 from P. sojae by a functional screening assay based on its wide range of cell death suppression activities. This effector gene was then introduced into the model plant Nicotiana benthamiana under control of the Cauliflower mosaic virus (CaMV) 35S promoter. PsCRN161-transgenic plants were similar to the wild type (WT) in growth and development, but had improved tolerance to Phytophthora infection and drought/salt stresses by up-regulating many defense-related genes. The results suggest that oomycete effectors could be used to enhance tolerance to biotic and abiotic stresses.

Results

Generation of transgenic N. benthamiana overexpressing PsCRN161

To screen effectors that protect plants from cell death induced by elicitors, we tested 23 P. sojae CRN effectors in N. benthamiana. The cell death inducers include P. sojae avirulence homolog 241 (Avh241) (Yu et al. 2012), necrosis-inducing protein PsojNIP (Qutob et al. 2002), CRN effector PsCRN63 (Liu et al. 2011), the R/AVR pair R3a (Huang et al. 2005) and P. infestans AVR3a (Armstrong et al. 2005), and mouse pro-apoptotic protein Bax (Lacomme and Cruz 1999). As shown in Supplementary Table S1, PsCRN161 and PsCRN70 exhibit strong cell death suppression activity and suppress cell death induced by all five elicitors tested, whereas other tested effectors do not. To elucidate the function of the newly identified effector PsCRN161, we generated transgenic N. benthamiana plants overexpressing green fluorescent protein (GFP):PsCRN161 fusions. As negative controls, transgenic lines expressing the GFP gene were produced. A total of 21 independent PsCRN161-expressing lines were obtained using kanamycin resistance selection. Most of the PsCRN161-transgenic lines showed strong fluorescent signal by fluorescent microscopy (Supplementary Fig. S1), indicating that the transgene was strongly expressed in transgenic lines. Therefore, we randomly selected four transgenic lines (#8, #14, #25 and #29) for further characterization. The four selected PsCRN161-transgenic lines showed 3 : 1 segregation for kanamycin resistance in the T₁ generation, implying that these lines contain a single transgene (Supplementary Table S2). Transgene integration and expression in four independent PsCRN161 lines was confirmed by genomic PCR (Supplementary Fig. S2A) and RT-PCR (Supplementary Fig. S2B). Western blot analysis using anti-GFP antibody further confirmed that the GFP:PsCRN161 fusion protein was successfully expressed at the expected size in transgenic lines (Fig. 1A). The PsCRN161-transgenic plants appeared to be similar to WT N. benthamiana and GFP-transgenic lines in terms of their vegetative and reproductive growth as well as morphological characteristics (Fig. 1B–D; Supplementary Fig. S2C–F), indicating that PsCRN161 expression does not considerably influence the development of N. benthamiana under normal growth conditions. The homozygous T₂ lines were selected on Murashige and Skoog (MS) medium containing 100 mg l^–1 kanamycin and further confirmed by observation of the fluorescent signal. These homozygous T₂ lines were used for further characterization.

Fig. 1

Overexpression of PsCRN161 has no visible effect on N. benthamiana. (A) Western blot analysis of the GFP control plants and T₁ transgenic lines (#8, #14, #25 and #29) using anti-GFP monoclonal antibodies. The asterisks indicate the expected size of the target proteins. PS, Ponceau staining. (B) Normal growth of the wild-type (WT) and the transgenic plants at 5 weeks after seed germination. (C and D) Fresh weight (C) and shoot height (D) of the plants. Dunnett’s test was used to compare the means of fresh weight and shoot height of the PsCRN161-transgenic plants and the control plants (P < 0.01). (E) Subcellular localization of GFP:PsCRN161 fusion protein in transgenic N. benthamiana. Leaf tissues were stained with DAPI to indicate plant nuclei. The photographs were taken using a confocal laser scanning microscope with excitation 488 nm for GFP and 405 nm for DAPI. The scale bar indicates 50 µm.

The majority of CRNs are localized to plant cell nuclei (Schornack et al. 2010, Stam et al. 2013). We analyzed whether PsCRN161 contains a nuclear localization signal (NLS) using cNLS Mapper software (Kosugi et al. 2009) and identified two monopartite NLSs in PsCRN161: the first one from 161 to 170 (ADKKRKRYWH) and the second one from 384 to 393 (HQPLKRLKLS). To examine further the subcellular localization of PsCRN161, we observed the green fluorescent signals in transgenic plant leaf tissues under a confocal laser scanning microscope. We found that GFP fluorescent signal was equally distributed in the cytoplasm and nucleus in GFP-transgenic plants. In contrast, the signals from GFP:PsCRN161 were mostly distributed in the nucleus (Fig. 1E). These results indicate that PsCRN161 is a nuclear-localized effector and further support that PsCRN161-transgenic plants were successfully obtained.

Cell death suppression activity of PsCRN161 in N. benthamiana

To verify the cell death suppression activity of PsCRN161 in stable transgenic N. benthamiana, we performed an in vivo cell death suppression assay using an Agrobacterium-mediated transient expression method (Torto et al. 2003). The cell death-inducing elicitors including Avh241, PsojNIP, PsCRN63, AVR3a/R3a and Bax were transiently expressed in PsCRN161- and GFP-transgenic lines. Similar to PsCRN161 alone (Supplementary Fig. S3), GFP:PsCRN161 fusions can suppress cell death triggered by all the tested elicitors in N. benthamiana whereas GFP cannot (Fig. 2A). Western blot analysis further confirmed that all the cell death inducers were expressed at similar levels in PsCRN161- and GFP-transgenic lines (Fig. 2B). These results confirm that GFP:PsCRN161 fusions did not affect the proper role of the effector, and PsCRN161 functions as a universal suppressor of many cell death-inducing elicitors.

Fig. 2

Cell death induced by elicitors was suppressed in the PsCRN161-transgenic plants. (A) Suppression of cell death induced by elicitors in PsCRN161-transgenic N. benthamiana. Agrobacterium GV3101 cells harboring the corresponding constructs were infiltrated into leaves of the transgenic plants. The photographs were taken at 6 d after infiltration. The numbers in each circled area represent the number of infiltrated area(s) exhibiting cell death over the total number of infiltrated leaf areas. (B) Confirmation of elicitor expression by Western blot. Anti-HA monoclonal antibodies were used to detect expression of HA-tagged cell death-inducing elicitors, and the expected band for each protein is indicated with an asterisk. PS, Ponceau staining.

Enhanced resistance to Phytophthora pathogens in PsCRN161-transgenic plants

Since programmed cell death (PCD) plays an important role in plant immunity, we evaluated the role of PsCRN161 in plant defense responses by inoculating the transgenic N. benthamiana leaves. All the inoculated leaves displayed water-soaked lesions at 36 h post-inoculation (Fig. 3A). However, the lesion diameters in PsCRN161-transgenic plants were significantly smaller than those in GFP-transgenic lines (Fig. 3A, B). Microscopic observations further showed that expression of PsCRN161 did not inhibit the germination of the cysts, and the pathogen can also develop appressorium-like structures 6 h post-inoculation (Supplementary Fig. S4), which facilitate penetration. However, a lower hyphal density was observed in the PsCRN161-transgenic plants compared with that of the GFP-transgenic plants 24 h post-inoculation (Supplementary Fig. S4). Moreover, fewer sporangia were produced on the surface of the leaves of the PsCRN161 line compared with the GFP line (Supplementary Fig. S4). Taken together, these observations suggest that expression of PsCRN161 inhibited colonization of Phytophthora pathogens.

Fig. 3

PsCRN161 expression increases plant resistance to two oomycete pathogens. (A) Typical leaves after infection with P. capsici mycelial plugs. The photographs were taken at 36 h after inoculation. (B) Lesion diameters of the infected N. benthamiana leaves. Bars indicate the SE from at least 12 independent biological replicates (**P < 0.01, Dunnett’s test). (C) The phenotypes of the whole plants inoculated with P. capsici zoospores. The typical photographs were taken at 4 d after inoculation. (D) Survival rates of the transgenic plants infected with P. capsici zoospores. Data were collected at 4 d after inoculation (**P < 0.01, Dunnett’s test). (E) Leaves infected with P. parasitica mycelial plugs. The data below indicate the average lesion diameters (cm) ± SE (**P < 0.01, Dunnett’s test). (F) Phenotypes of transgenic plants infected with P. parasitica zoospores. The data indicate the survival rates of the plants at 4 d after inoculation (**P < 0.01, Dunnett’s test).

We then inoculated the whole seedlings with zoospores using the root-dip method. The bottom leaves of both the PsCRN161- and GFP-transgenic plants showed symptoms of wilting and leaf etiolation (Fig. 3C). However, the upper leaves of the PsCRN161-transgenic plants showed no significant wilting symptoms compared with the GFP-transgenic lines. In addition, more GFP-transgenic plants fell over compared with the PsCRN161-transgenic lines after inoculation. We defined plants with both wilting and falling over symptoms as dead plants. The survival rate was significantly higher in the PsCRN161-expressing plants than in the GFP lines (Fig. 3D). Similar results were obtained when we inoculated the transgenic plants with another oomycete pathogen, P. parasitica (Fig. 3E, F). The PsCRN161-transgenic N. benthamiana showed smaller lesion diameters (Fig. 3E) and higher survival rates (Fig. 3F) after infection with P. parasicita, compared with the GFP lines, indicating that expression of PsCRN161 enhanced plant resistance to P. parasitica. Taken together, expression of PsCRN161 improves plant resistance to the tested oomycete pathogens.

Increased salt tolerance in PsCRN161-transgenic plants

PCD is proved to occur in response to abiotic stresses such as salinity, drought and cold stresses (Shabala 2009, Duan et al. 2010). Since PsCRN161 suppresses cell death triggered by a wide range of cell death inducers, we evaluated whether the expression of PsCRN161 influences plant tolerance to abiotic stresses including salt and drought stresses. To test the influence of PsCRN161 expression on seed germination in response to salt stress, plant seeds were germinated on MS plates supplemented with different concentrations of NaCl. No significant differences in germination rates were observed between the PsCRN161-transgenic lines and control plants without NaCl treatment (Fig. 4A). However, when the concentration of NaCl was increased to 100 mM, the germination rate of PsCRN161-transgenic seeds was significantly higher than that of WT and GFP-transgenic plants (Fig. 4A, B). A similar result was obtained when the concentration of NaCl was further increased to 150 mM (Fig. 4A, B).

Fig. 4

PsCRN161 expression enhances salt tolerance in N. benthamiana. (A) Germination of N. benthamiana seeds in the presence of NaCl (0, 100 and 150 mM). The photographs were taken at 12 d after sowing. (B) Germination rates of transgenic N. benthamiana seeds under NaCl treatments. The rates were scored within 12 d after sowing. Bars represent the SE. (C) Root lengths and (D) fresh weight of the transgenic N. benthamiana under NaCl treatment. The seeds were sown on MS medium for 3 d, and the radicles were transferred to MS medium containing different concentration (0, 100 and 150 mM) of NaCl. Bars represent the SE (**P < 0.01, Dunnett’s test). (E) The representative phenotypes of transgenic plants and control plants under salt treatment. Three-week-old plants were treated with 150 mM NaCl for 2 weeks. (F) Shoot height of the transgenic plants and control plants in (E) (**P < 0.01, Dunnett’s test). (G) Survival rates of the transgenic plants under salt treatment. Eight-week-old plants were treated with 150 mM NaCl for 2 weeks. Bars represent the SE (**P < 0.01, Dunnett’s test). The typical treated plants are shown in Supplementary Fig. S5.

To confirm the increased salt tolerance, the post-germination growth of the transgenic plants was analyzed. The plant seeds were allowed to germinate on MS plates for 4 d and then transferred onto MS plates containing different concentrations of NaCl. There was no significant difference in root length between the PsCRN161-transgenic lines and the controls treated with 0 mM NaCl. However, the root length was significantly longer compared with the controls in the presence of 100 or 150 mM NaCl (Fig. 4C; Supplementary Fig. S5). Accordingly, the fresh weight was significantly higher in the PsCRN161 lines than that in the WT and GFP-transgenic lines when grown on 100 or 150 mM NaCl (Fig. 4D). To examine the salt tolerance further, plants were grown under salt treatment conditions (0 and 150 mM NaCl) in soil. After 2 weeks, all plants showed similar growth without salt treatment (Fig. 4E). In contrast, the growth of the PsCRN161-transgenic plants was considerably faster compared with that of the WT and GFP-transgenic plants under 150 mM NaCl treatment (Fig. 4E). The shoot height of the PsCRN161 lines was also significantly higher than that of the GFP-transgenic plants under salt stress (Fig. 4F). In the reproductive stage, >80% of the control plants showed complete leaf wilting and withering symptoms 2 weeks after 150 mM NaCl treatment (Fig. 4G; Supplementary Fig. S6), and we define these as dead plants, whereas >70% of the PsCRN161-transgenic plants survived under the same conditions (Fig. 4G; Supplementary Fig. S6). These results suggest that expression of PsCRN161 improves the salinity tolerance in N. benthamiana.

Increased drought tolerance in PsCRN161-transgenic plants

To investigate the effects of PsCRN161 expression on plant drought tolerance, plant seeds were sown onto MS medium containing different concentrations of mannitol (100 and 200 mM) to mimic osmotic stress. As shown in Fig. 5A, mannitol treatment significantly delayed the germination of N. benthamiana seeds compared with the control. However, the germination rate of PsCRN161-transgenic seeds was significantly higher than that of the WT and GFP-transgenic lines under treatment with 100 or 200 mM mannitol (Fig. 5A). To confirm further the plant drought tolerance at the vegetable growth stage, water was withheld from the plants for 12 d. After 12 d of drought treatment, WT and GFP-transgenic plants completely wilted, whereas PsCRN161-transgenic plants were less affected (Fig. 5B). Moreover, under drought stress, GFP-transgenic and WT plants showed a mild decrease in growth compared with the PsCRN161-transgenic plants (Fig. 5B). When they were watered after the drought treatment, the PsCRN161-transgenic plants recovered faster than the GFP-transgenic and WT plants (Fig. 5B). In addition, the rate of water loss from detached leaves of control plants was higher than that of PsCRN161-transgenic plants under drought conditions (Fig. 5C), and the PsCRN161-transgenic plants displayed higher survival rates (Fig. 5D). These results indicate that expression of PsCRN161 improves the drought tolerance in N. benthamiana.

Fig. 5

PsCRN161 expression enhances drought tolerance in transgenic plants. (A) Germination rates of transgenic plant seeds under treatment with mannitol. The seeds were sown on MS medium supplemented with different concentrations of mannitol (0, 100 and 200 mM). The rates were calculated and recorded within 12 d. Bars represent the SE (**P < 0.01, Dunnett’s test). (B) Phenotypes of PsCRN161-transgenic plants under drought stress. Eight-week-old plants were not watered for 12 d. They were then watered for 1 d to allow them to recover. (C) Water loss from the detached leaves. The water loss was defined as the loss of fresh weight in the samples. Bars represent the SE. (D) Survival rates of the transgenic plants under drought stress. The plants were treated for 15 d. Bars represent the SE (**P < 0.01, Dunnett’s test).

Up-regulation of many defense-related genes in PsCRN161-transgenic plants

To elucidate preliminarily the mechanisms through which biotic and abiotic stress tolerance is enhanced in PsCRN161-transgenic plants, digital gene expression (DGE) tag profiling was conducted in leaf tissues of 8-week-old plants under normal growth conditions. Ten housekeeping genes (Liu et al. 2012) were used to evaluate the DGE data, and the relative fold changes (the PsCRN161 line vs. the GFP line) of the tested genes were around 1.0 (Supplementary Table S3), indicating that the expression levels of the housekeeping genes were not significantly different between the PsCRN161- and GFP-transgenic lines. This result indicates that the DGE data were reliable, and suitable for further analyses. After statistical analysis, 75 and 21 unigenes were significantly up-regulated and down-regulated, respectively, in PsCRN161-expressing N. benthamiana compared with the GFP line using a 2-fold cut-off (Supplementary Table S4). Many defense-related genes were up-regulated in PsCRN161-transgenic plants compared with GFP plants (Supplementary Table S5). Enhanced expression of six ABC transporter genes was seen, which may be involved in responses to biotic and abiotic stresses. A significant up-regulation of two Cyt P450 genes was observed in PsCRN161-transgenic plants, suggesting that Cyt P450 may play a role in stress responses. Nine receptor-like kinases (RLKs) were significantly up-regulated in PsCRN161-expressing plants, indicating that PsCRN161 expression may considerably influence plant cell signaling cascades. To validate the results obtained by DGE analysis, quantitative reverse transcription–PCR (qRT–PCR) was performed. Expression levels of two ABC transporter-encoding genes (NbS00001556g0001.1 and NbS00001556g0005.1), two Cyt P450 genes (NbC24805505g0002.1, NbC25673618g0001.1), two RLKs (NbS00055677g0008.1 and NbS00031824g0005.1), a late blight resistance protein homolog R1a-4 (NbS00033032g0022.1) and a pathogen-induced calmodulin-binding protein (NbS00055701g0010.1) were determined. The expression patterns measured by qRT–PCR were highly consistent with the DGE results (Fig. 6). To evaluate preliminarily the role of the up-regulated genes in response to biotic stress, we detected the expression levels of three RLKs (NbS00033954g0001.1, NbS00023927g0006.1 and NbS00031824g0005.1) and two Cyt P450s (NbC24805505g0002.1 and NbC25673618g0001.1) at 12 h post-inoculation. The qRT–PCR result showed that all the selected genes were strongly induced by P. parasitica (Supplementary Table S6), suggesting that they contribute to plant resistance against Phytophthora pathogen.

Fig. 6

Validation of DEGs by quantitative PCR. Relative gene expression levels (fold change) in PsCRN161-transgenic lines compared with GFP lines were determined using qRT–PCR. Three individual qRT–PCR analyses were performed from three biological replicates. The results displayed similar trends from these individual qRT–PCR analyses. A representative result is presented here. Error bars represent the SE from three technical repeats. Nicotiana benthamiana EF1α was used as a reference gene. The relative expression levels were calculated using the delta-delta Ct method.

Discussion

By using a functional screening assay based on cell death suppression activity, we identified PsCRN161 from P. sojae and found that it suppresses cell death triggered by the tested elicitors, and PsCRN161 expression in N. benthamiana enhances tolerance to biotic and abiotic stresses. It targets the plant nucleus to regulate expression of many defense-related genes, and then considerably modifies plant physiology and cell function to confer resistance. Therefore, we suggest that oomycete effectors could be directly used to improve plant tolerance to biotic and abiotic stresses.

Phytophthora spp. are hemibiotrophic pathogens with biotrophic action during early infection and necrotrophic action in the later stage. CRNs were initially identified as crinkling- and necrosis-inducing proteins, and were once considered to contribute mainly to necrotrophic stages of infection (Torto et al. 2003). However, recent studies demonstrated that expression of CRNs rarely triggered cell death (Shen et al. 2013, Stam et al. 2013). Moreover, we showed previously that many CRNs suppressed cell death induced by elicitors such as the PAMP (pathogen-associated molecular pattern) molecule INF1 and the R/AVR pair R3a/AVR3a (Shen et al. 2013, Rajput et al. 2014), suggesting that CRNs may play distinct roles in pathogenesis. The spectra of cell death suppression differ between CRN members (Shen et al. 2013), indicating that CRNs target different plant components to exert biological functions. A similar phenomenon has been found in some oomycete RxLR effectors in which the spectra of cell death also differ between RxLRs, suggesting that these effectors may co-operate to regulate plant cell death during infection (Wang et al. 2011). Similar to PsCRN161, RxLR effectors can also function as broad-spectrum cell death suppressors, and many members of RxLRs suppress cell death triggered by either INF1, Bax or effectors (Wang et al. 2011). However, the cell death suppression mechanisms of these RxLRs and their roles in pathogenicity are still unknown.

Bax inhibitor-1 (BI-1) is a broad-spectrum cell death suppressor, and it is conserved in animals and plants (Ishikawa et al. 2011). Expression of plant BI-1 attenuates cell death induced by biotic stresses (pathogens) and abiotic stresses such as heat, cold, salt and drought, and chemical-induced oxidative stresses (Ishikawa et al. 2011). BI-1 is an endoplasmic reticulum (ER)-resident transmembrane protein and interacts with multiple proteins to alter intracellular Ca²⁺ flux control and lipid dynamics. The level of BI-1 protein is considered as a rheostat to regulate the threshold of ER stress-induced cell death (Ishikawa et al. 2011). Unlike BI-1, PsCRN161 is a nuclear-localized protein, suggesting that it uses a different mechanism to regulate cell death. Identification and characterization of PsCRN161-interacting proteins will be useful to elucidate the mechanism underlying broad-spectrum cell death suppression of PsCRN161. The broad-spectrum cell death suppressors may target a highly conserved ‘core’ mechanism and serve as useful probes to elucidate cell death signaling in plants.

Cell death plays a central role in innate immunity in both animals and plants (Coll et al. 2011). It has been documented that BI-1 is involved in plant defense response, as well as the HR, a well-characterized form of PCD (Ishikawa et al. 2011). Overexpression of barley BI-1 leads to hypersusceptibility to the biotrophic pathogen Blumeria graminis, and the cell death suppression function of BI-1 benefits B. graminis to infect host plant cells by regulating the capacity of cell wall-associated defense responses in barley (Ishikawa et al. 2011). We showed previously that a P. sojae CRN effector (PsCRN70) exhibits broad-spectrum cell death suppression activity, and PsCRN70 expression in plants elicits plant defense responses and promotes growth of hemibiotrophic Phytophthora pathogens (Rajput et al. 2014). In contrast, here we showed that PsCRN161 expression increased resistance to Phytophthora infection. These results suggest that PsCRN161 uses a different mechanism to modulate plant immunity.

We found that overexpression of PsCRN161 enhanced plant tolerance to Phytophthora pathogen infection, salt and drought stresses. Genome-wide gene expression analysis showed that many defense-related genes were up-regulated in PsCRN161-transgenic N. benthamiana compared with GFP lines. Among them, five ABC transporter genes were significantly up-regulated in PsCRN161-expressing plants. It has been shown that ABC transporters are involved in responses to biotic and abiotic stresses (Campbell et al. 2003, Stein et al. 2006, Kim et al. 2010). Up-regulation of two Cyt P450 genes was observed in PsCRN161-transgenic plants. Cyt P450 genes were also shown to contribute to resistance against biotic and abiotic stresses (Narusaka et al. 2004a, Narusaka et al. 2004b). AtCYP76C2 was found to be up-regulated by several biotic and abiotic stresses, such as Alternaria brassicicola infection, drought, high salinity and UV stress (Narusaka et al. 2004b). Nicotiana benthamiana P450 (NbC24805505g0002.1) is a homolog of AtCYP76C4, which belongs to the same subfamily as AtCYP76C2. The two Cyt P450 genes up-regulated in PsCRN161-transgenic plants are also homologs of the Cyt P450 (AtCYP81D8) (Narusaka et al. 2004a). Similarly, the expression of AtCYP81D8 was induced upon different biotic and abiotic stresses (Narusaka et al. 2004a). These results implied that the two Cyt P450 genes identified from this study are also involved in response to biotic and abiotic stresses. Interestingly, enhanced expression of nine RLKs was observed in PsCRN161-expressing plants. Plant RLKs are transmembrane proteins with N-terminal extracellular domains and C-terminal intracellular kinase domains. RLKs control a wide range of biological processes, including development, disease resistance and abiotic stress tolerance (Shiu and Bleecker 2001, Marshall et al. 2012). From these nine RLKs, we have identified NbS00023927g0006.1 as a homolog of FLS2 and NbS00033954g0001.1 as a homolog of SOBIR1/EVR. FLS2 is an important pattern recognition receptor involved in pattern-triggered immunity (Nicaise et al. 2009), and SOBIR1/EVR interacts with receptor-like proteins to regulate plant immunity against fungal pathogens (Liebrand et al. 2013). We conceived that increased expression of these defense-related genes may confer resistance to biotic and abiotic stresses in PsCRN161-transgenic plants. Interestingly, we showed previously that expression of another CRN effector, PsCRN115, also enhanced N. benthamiana resistance to biotic and abiotic stresses. However, the up-regulated genes in the PsCRN115-transgenic plants are dominant in genes encoding heat shock proteins and Cyt P450s (Zhang et al. 2015b). These results indicate that the two CRN effectors may regulate plant resistance to stresses by distinct mechanisms.

Similar to other CRNs reported, PsCRN161 was localized to the plant nucleus. The nucleus is considered as the ‘command center’ of the cell, and governs gene expression and controls whole-cell physiology. A growing number of studies indicate that bacterial pathogens can deliver effectors to the host nucleus to subvert host nuclear processes by directly interfering with transcription, chromatin remodeling or DNA repair (Bierne and Cossart 2012). The TAL effectors enter the host nucleus, bind DNA sequences and turn on host gene expression. Besides the TAL effectors, bacterial pathogens produce effectors to target the nucleus and manipulate a variety of host nuclear processes (Bierne and Cossart 2012). These effectors are termed ‘nucleomodulins’. The majority of CRNs tested are localized to the plant nucleus. PsCRN161 may function as a nucleomodulin and target the plant nucleus to regulate host gene expression and cause long-term epigenetic effects on the host. However, it remains unclear whether PsCRN161 regulates expression of genes identified by DGE directly by binding to host DNA sequences or indirectly by affecting other regulators, such as transcription factors or chromatin modifiers.

In conclusion, we showed that the expression of an oomycete effector, PsCRN161, improved plant tolerance to biotic and abiotic stresses without leading to developmental changes. Constitutive expression of some effectors in plants may considerably modify plant physiology and cell function, and therefore confer plant resistance to stresses.

Materials and Methods

Plant material and growth conditions

Nictiana benthamiana seeds were placed on MS medium for germination. Seedlings about 2–3 cm long were transferred to soil pots and grown in a greenhouse chamber at 25°C under a 16/8 h light/dark cycle.

Agrobacterium-mediated transient gene expression

Agrobacterium tumefaciens strain GV3101 containing the corresponding constructs were cultured in Luria–Bertani medium at 28°C with shaking at 220 r.p.m. for 48 h. Bacterial cells were harvested and washed three times with 10 mM MgCl₂, and resuspended in infiltration buffer (10 mM MgCl₂, 10 mM MES, pH 5.6, and 150 mM acetosyringone) to an OD₆₀₀ of 0.4 before infiltration into N. benthamiana leaves with a needless syringe.

Plasmid constructs and N. benthamiana transformation

The PsCRN161 gene (accession number: XM_009523001) was amplified with the corresponding primers (primer information in Supplementary Table S7), and cloned into the pBinGFP2 vector with BamHI and XbaI. The recombinant plasmid was confirmed by sequencing and introduced into A. tumefaciens strain EHA105 by the electroporation method. Nicotiana benthamiana transformation was conducted by Agrobacterium-mediated leaf disc transformation as reported previously (Rajput et al. 2014).

Protein extraction and Western blot

Nicotiana benthamiana leaf tissues were ground in liquid nitrogen, and protein was extracted with protein extraction buffer (50 mM HEPES-KOH, pH 7.5, 150 mM KCl, 1 mM EDTA, 0.5% Triton X-100, 1 mM dithiothreitol and 1 × protease inhibitor cocktail). Suspensions were mixed and centrifuged at 12,000 × g for 15 min at 4°C. The supernatant was mixed with SDS loading buffer. The proteins were denatured and separated on a 12% SDS–polyacrylamide gel. Proteins were then transferred from the gel to an Immobilon-P^SQ polyvinylidene difluoride membrane. Membranes were probed with mouse monoclonal anti-GFP or anti-HA antibodies (Sigma-Aldrich) at a dilution of 1 : 3,000. IRDye 800CW-conjugated goat anti-mouse IgG second antibodies were diluted with 5% (m/v) non-fat milk in PBST [phosphate-buffered saline with 0.1% (v/v) Tween-20]. Membranes were visualized using a LI-COR Odyssey scanner with excitation at 700 and 800 nm.

Confocal microscopy

To observe the subcellular localization of the GFP:PsCRN161 fusion protein, 5 µg ml^–1 4′,6-diamidino-2-phenylindole (DAPI) solution was infiltrated into transgenic N. benthamiana leaves. The infiltrated plant leaves were cut into small squares and mounted in water under a coverslip. Fluorescence and DAPI staining were visualized with a Zeiss LSM710 confocal microscope. The excitation wavelength for GFP was 488 nm, and it was 405 nm for DAPI. The GFP-transgenic N. benthamiana leaves were used as controls. Images were processed using the Zeiss LSM710 software.

Phytophthora infection assays

Phytophthora parasitica strain Pp025 and P. capsici strain Pc35 were grown on 10% (v/v) V8 juice agar at 25°C in the dark. For detached leaves, mycelial plugs were applied onto the abaxial side of detached N. benthamiana leaves and disease symptoms were recorded at 36 h after inoculation. For whole seedlings, plants were inoculated using the root-dip inoculation method. The whole transgenic plants were inoculated with P. capsici or P. parasitica zoospores. The disease symptoms were monitored for 10 d. Dunnett’s test was used for statistical analysis (P < 0.01). The survival rates were calculated by dividing the overall survival after infection by the survival as observed after mock treatment.

Salt and drought stress analyses

For salt treatment, seeds were germinated in MS medium supplemented with different concentrations of NaCl (0, 100 and 150 mM). To evaluate the effect of PsCRN161 expression on post-germination growth, the seeds were sown on MS medium for 4 d and then the radicles were transferred onto MS medium with different concentrations of NaCl, and root elongation was monitored and measured. To evaluate the effect of salt stress on plant growth in soil, 3-week-old plants were grown under salt treatment (0 and 150 mM NaCl) for 2 weeks, and the shoot height was measured. To examine the salt tolerance of the transgenic plants in the reproductive stage, 8-week-old plants grown in soil were irrigated with 150 mM NaCl for 2 weeks. The plants showing complete leaf wilting and withering symptoms after NaCl treatment were defined as dead plants. The survival rates were calculated by dividing the overall survival after salt treatment by the survival as observed after mock treatment. For drought treatment, seed germination rates were calculated by counting the germination percentage on MS medium supplemented with mannitol (0, 100 or 200 mM). To test the drought tolerance of transgenic plants in soil, water was withheld completely from 8-week-old plants for 12 d, and then they were watered and allowed to recover for 1 d. For water loss assay, leaves of PsCRN161-transgenic plants and control lines were detached and incubated at 37°C and weighed at the indicated time points. The rate of water loss was defined as the loss of fresh weight of the samples. Salt and drought treatment experiments were repeated at least three times. Dunnett’s test was used for statistical analysis (P < 0.01).

Digital gene expression profiling analysis

Total RNA was extracted from 6-week-old N. benthamiana leaf tissues with an RNAsimple Total RNA Kit (Tiangen). RNA quality was assessed by the optical density at 260 nm/optical density at 280 nm. RNAs from four biologically independent replicates were mixed with equal quantities for tag preparation. Illumina sequencing was conducted using a Hiseq2000 to produce 100 bp paired-end data. Clean reads were mapped to N. benthamiana reference gene sequences using Tophat with default parameters. The N. benthamiana reference gene sequences were downloaded from the database (http://solgenomics.net/organism/Nicotiana_benthamiana/genome). The expression level of each gene was quantified and normalized using the reads per kilobase per million mapped reads (RPKM) method. Differentially expressed genes (DEGs) were defined by calculating the statistical significance using GFOLD software (Feng et al. 2012) [GFOLD > 1 or GFOLD less than –1; log2 (fold change) > 2 or log2 (fold change) less than –2]. Functional annotation analysis of DEGs was performed by BLAST against the NCBI NR database with an E-value cut-off of 10^–5.

Quantitative RT–PCR validation

Functionally important and representative DEGs were selected for validation using qRT–PCR. Gene-specific primers for each DEG were designed, and are listed in Supplementary Table S4, and the N. benthamiana EF1α gene was used as a reference. qRT–PCR was performed using a SYBR® Premix ExTaq II reagent kit (TAKARA). The relative expression level of each gene was determined by using the delta-delta Ct method.

Funding

This work was supported by the National Science and Technology Major Projects [grant No. 2014ZX0800910B); the National Natural Science Foundation of China (NSFC) [grant Nos. 31301613 and 31371894]; the Chinese Ministry of Education [Key project no. E200909]; Nanjing Agricultural University (NJAU) [Youth Science and Technology Innovation Fund (KJ2013005)].

Abbreviations

Abbreviations
 
• Avh241

  avirulence homolog 241

 
• BI-1

  Bax inhibitor-1

 
• CRN

  Crinkler

 
• DAPI

  4′,6-diamidino-2-phenylindole

 
• DEG

  differentially expressed gene

 
• DGE

  digital gene expression

 
• HR

  hypersensitive response

 
• ETI

  effector-triggered immunity

 
• GFP

  green fluorescent protein

 
• MS

  Murashige and Skoog

 
• NIP

  necrosis-inducing protein

 
• NLS

  nuclear localization signal

 
• PCD

  programmed cell death

 
• qRT–PCR

  quantitative reverse transcription–PCR

 
• RLK

  receptor-like kinase

 
• ROS

  reactive oxygen species

 
• WT

  wild type

Acknowledgements

We thank Dr. Weixing Shan (Northwest A&F University) for providing P. parasitica strain Pp025, and Brett Tyler (Oregon State University) for comments on the manuscript.

Disclosures

The authors have no conflicts of interest to declare.