Functional Analysis of KPvRxLR27, a Novel Plasmopara viticola Effector from a Korean Isolate, and Its Role in Hypersensitive Response

Article information

Plant Pathol J. 2025;41(1):28-37
Publication date (electronic) : 2025 February 1
doi : https://doi.org/10.5423/PPJ.OA.09.2024.0141
Department of Applied Biology, Chungnam National University, Daejeon 34134, Korea
*Corresponding author. Phone) +82-42-821-5762, FAX) +82-42-823-8679, E-mail) sangkeun@cnu.ac.kr
Handling Editor : Hee Jin Park
Received 2024 September 11; Revised 2024 November 25; Accepted 2024 November 27.

Abstract

Plasmopara viticola causes grape downy mildew, one of the most notorious diseases of cultivated grapes that damage vineyards worldwide. The pathogen secretes various effector molecules to infect and modulate the host biological processes. In this study, we aimed to evaluate the roles of KPvRxLR27, an arginine–any amino acid–leucine–arginine (RxLR) effector isolated from P. viticola JN-9 from Jeonju (South Korea) with respect to the reported Bcl-2-associated X and inverted formin1in inducing cell death in non-host Nicotiana benthamiana and resistant grape host cultivars via Agrobacterium-mediated transient transformation. We found that, KPvRxLR27 induced programmed cell death in N. benthamiana and rapid hypersensitive response in resistant grape cultivars. Agroinfiltration assay revealed that putative N-glycosylation at the N186 amino acid sequence and nuclear localization signal motifs at the C-terminus were critical for the effector’s cell death-inducing activity of KPvRxLR27. Overexpression assay revealed that KPvRxLR27 was abundantly expressed in the plasma membrane and nuclear regions and activated the accumulation of reactive oxygen species in N. benthamiana. Moreover, KPvRxLR27 expression was significantly delayed in the resistant cultivar than in the susceptible cultivar. Our results suggest KPvRxLR27 as a potential avirulence gene recognized by the host receptors to activate the host immune response-associated genes, providing valuable insights to enhance the pathogen resistance of commercial cultivars.

Owing to the continuous evolution of pathogenic microorganisms, plants have developed a two-layer innate immunity to survive and restrict pathogen infections (Chisholm et al., 2006; Jones and Dangl, 2006). The first layer of this immunity is the pathogen-associated molecular pattern (PAMP) triggered immunity (PTI) that recognizes microbe-conserved molecules known as PAMPs or microbe-associated molecular patterns via pattern recognition receptor molecules; the most commonly known PAMPs include, Phytophthora infestans inverted fomin1 (INF1) and bacterial flagellin (Ngou et al., 2021). Recognition of such molecular patterns via PTI triggers signaling cascades for reliable defense responses including defense-related genes expression, callose deposition, and reactive oxygen species accumulation for oxidative burst (Dodds and Rathjen, 2010). However, some pathogens such as oomycetes, interfere with PTI by secreting effector molecules for the establishment of effector-triggered susceptibility. The second layer of immunity is mediated by resistance (R) proteins recognizing pathogen effectors to induce a steadier defense and limit the spread of infection in resistant cultivars; which is termed as effector-triggered immunity (ETI), induces a rapidly localized hypersensitive response (HR) (Pruitt et al., 2021).

Recent studies on oomycetes have focused on the identification and functional verification of R gene-recognizing effectors known as avirulence (Avr) genes and elucidation of the mechanisms underlying the interactions of Avrblb2, in Phytophthora infestans (Oh et al., 2009), and PvRxLR16 and PvAvh74 in Plasmopara viticola (Xiang et al., 2017; Yin et al., 2019) with plant R genes.

Plasmopara viticola is among the most notorious biotrophic oomycetous microorganisms infecting almost all green parts of grape to cause the downy mildew disease. During infection, the pathogen secretes various effector molecules from haustoria into the inter-cellular (apoplastic effectors) as well as intracellular (cytoplasmic effectors) spaces to modulate the host biological processes and establish infection (Bozkurt et al., 2012). Genome sequencing has facilitated the identification of many P. viticola effectors (Yin et al., 2017). The most extensively studied cytoplasmic effectors of P. viticola are arginine–any amino acid–leucine–arginine (RxLR) effectors containing a conserved N-terminal signal peptide preceding the RxLR motif, which exhibit two main functions. These effectors mainly suppress programmed cell death while a small proportion of these effectors trigger cell death in different host compartments. For example, seven of the 26 candidates from a highly virulent P. viticola strain YL which were predominantly located in the nucleus triggered programmed cell death whereas 19 suppressed HR induced by Bcl-2-associated X (BAX) and INFI in Nicotiana benthamiana (Chen et al., 2020). The pro-apoptotic cell death promoting mouse Bcl-2 member protein (BAX) was reported to induce HR in N gene containing tobaco plants (Lacomme and Santa Cruz, 1999). Similally, Phytophthora infestans secreted INF1 triggers in planta defense-related HR following recognition by receptor-like protein ELICITIN RESPONSE in association with 1-ASSOCIATED KINASE1/SOMATIC EMBRYOGENESIS RECEPTOR KINASE 3 (BAK1/SERK3) proteins.

Out of the 83 PvRxLR effectors from P. viticola JL-7-2 predicted to target various cellular compartments such, as nucleus, cytoplasm, plasma membrane, and chloroplasts, only 10 triggered cell death in N. benthamiana (Liu et al., 2018). PvRxLR effectors require various plant cell components to induce cell death in host and non-host systems; EDS1, SGT1, RAR1 HSP90, and NDR1 are necessary for the P. viticola Avr homolog (PvAvh74) to induce cell death in N. benthamiana (Yin et al., 2019). However, due to the biotrophic nature of the pathogen and perennial nature of its host, the mechanisms underlying PvRxLR effector function during infection particularly in the Korean viticulture remain unknown. Therefore, comprehensive analyses of the biological functions of these effectors are important scientific for viticulture.

In this study, we aimed to analyze the function of the KPvRxLR27 effector from a Korean P. viticola JN-9 isolate. KPvRxL27 induced programmed cell death, in the non-host N. benthamiana and triggered rapid HR in resistant grape cultivars. KPvRxLR27 overexpression induced the accumulation of reactive oxygen species and cell death after nuclear localization in N. benthamiana. Moreover, transient expression analysis revealed that putative N-glycosylation site and nuclear localization signal (NLS) motifs at the C-terminus were indispensable for the cell death-inducing activity of KPvRxLR27. These findings provide insight into the mechanisms underlying P. viticola - grape interactions following infection thus facilitating the improvement of grape resistance to downy mildew disease in South Korea.

Materials and Methods

Plants, microbial strains, and growth conditions

Grape plants were cultivated using cuttings collected from the Rural Development Administration of South Korea. They were grown with tobacco plants (N. benthamiana) in the Chungnam National University growth chamber at 25°C under 16/8 h light/dark cycles. Escherichia coli DH5α was grown on Luria-Bertani medium which was used for cloning and amplification of recombinant plasmids at 37°C. Agrobacterium tumefaciens GV3101 used for in plant transient expression was cultured in yeast extract peptone (YEP) broth with an appropriate selection antibiotic at 28°C and P. viticola sporangia were grown on leaf discs excised from Cabernet sauvignon, a susceptible grape cultivar as described in our previous report (Semunyana et al., 2023).

DNA extraction and vector construction

Genomic DNA was extracted from the sporangia of P. viticola JN-9 isolate using cetyltrimethylammonium bromide method, as previously described (Lee et al., 1988). Signal peptide allows the secretion of effector from the oomycetes and the signal peptide sequence-free P. viticola effector candidates keep their cell death-inducing activity in plants (Kamoun, 2006; Yin et al., 2019). Therefore, the open reading frame of the KPvRxLR27 gene minus its predicted signal peptide sequence was) amplified from the genomic DNA of P. viticola JN-9 and the PCR product was digested using restriction enzymes and ligated into the pGR106 expression vector to construct pGR106–effector for functional analysis. For subcellular localization KPvRxLR27 vector with a green fluorescent protein (GFP) sequence was ligated into pCAMBIA1302 to construct pCAMBIA1302–effector construct. All the primer sets used in this study are listed in Supplementary Table 1. Diagrams depicting all vector constructs used in this study are shown in, Supplementary Fig. 1.

Agrobacterium-mediated transient transformation

Recombinant plasmids containing the confirmed sequences were introduced into A. tumefaciens GV3101 via liquid nitrogen flash freezing and heat shock. Positive clones were selected on YEP broth medium with rifampicin (50 μg/ml) and kanamycin (50 μg/ml) and cultured in liquid YEP broth with the above-mentioned antibiotics, in a shaking incubator at 28°C and 200 rpm. After 48 h of incubation, A. tumefaciens cells were harvested via centrifugation at 3,000 rpm for 10 min at 4°C, washed thrice with 10 mM MgCl2, and re-suspended in MMA infiltration buffer (20 g sucrose, 5 g MS salts without vitamin,10 mM MES [1.95 g], 2 ml NaOH [1 M] and 1 ml acetosyringone [200 mM] mixed in 1 liter of autoclaved distilled water, pH 5.6), The cell suspension was adjusted to OD600 of 0.5 and incubated at room temperature prior to infiltration. The 3rd to 6th leaves of grape cultivars as well as the leaves of 4–5 weeks old N. benthamiana plants were infiltrated using 1 ml needle less syringe. This agroinfiltration assay was conducted in triplicate.

Electrolytic leakage assay

Ion leakage was measured to quantify effector induced-cell death in N. benthamiana as previously described (Yin et al., 2019). Five leaf discs excised at the infiltrated sites by a cork borer (1 cm diameter) were rinsed thrice within 5 ml autoclaved distilled water, and incubated for 3 h at room temperature. Electrical conductivity of the solution was measured using seven direct pH/conductivity meters (SD23) to obtain the A value. The samples were boiled for 25 min, cooled and EC was measured to obtain the B value. Electrolytic leakage was measured in three independent experiments, and the average of both A and B values was considered for the calculation of electrolyte leakage as a percentage (%) of A values over B values.

Confocal microscopy and detection of oxidative burst

KPvRxLR27 gene without its signal peptide sequence was ligated into the pCAMBIA1302-GFP vector to obtain a plasmid containing the KPvRxLR27-GFP construct. After verifying the success of A. tumefaciens GV3101 transformation using the recombinant plasmid, KPvRxLR27 was introduced into N. benthamiana via Agrobacterium-mediated transient expression. Then at 48–72 h post infiltration, the infiltrated sites were excised, mounted on microscopic discs and the images were observed using a confocal microscope (Nikon Eclipse Ni, Tokyo, Japan), with the GFP excitation wavelength of 488 nm.

To detect reactive oxygen species (ROS) accumulation, we stained N. benthamiana leaves with 3,3′-diaminobenzidine (DAB) as previously described (Xiang et al., 2017). Curtly agro infiltrated N. benthamiana leaves were detached 72 h post infiltration and soaked in DAB solution (1 mg/ml autoclaved distilled water) overnight at 25°C. Following incubation, the leaf tissues were decolorized by boiling in 95% ethyl alcohol for 15 min and photographed using a Nikon Z50 (DX50-250). Experiments were performed in triplicate.

RNA extraction and reverse transcription polymerase chain reaction analysis

Leaf discs of grape susceptible (Cabernet sauvignon) and resistant (Moldova) cultivars were inoculated with P. viticola (4 × 104 sporangia/ml) suspension and incubated at 20°C and 16/8 h light/dark cycles. RNA was extracted at different time intervals and cDNAs 0.5–1 μg total RNA was reverse transcribed to cDNA with 20 μl total volume using the smart gene Compact cDNA synthesis kit (SmartGene, Seoul, Korea) following the manufacturer’s protocol. Vitis vinifera and P. viticola actin genes were used as references. In brief, PCR amplification involved pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 s, annealing at 58°C for 45 s, extension at 72°C for 1 min, and final extension at 72°C for 5 min. The resulting PCR products were visualized on 1% agarose gel electrophoresis. Sequence-specific primer sets designed for KPvRxLR27 full-length PCR amplification were also used for reverse transcription polymerase chain reaction (RT-PCR) and were listed in Supplementary Table 1.

Bioinformatic and Statistical analysis

Conserved amino acid motifs for subcellular localization were predicted using LOCALIZER (https://localizer.csiro.au/). Scan Prosite (https://prosite.expasy.org/scanprosite/) was used to predict the N-glycosylation sites. The means computed from different experimental treatments were recorded in Microsoft Excel and imported into SAS (v.9.1, SAS Institute Inc., Cary, NC, USA). Differences among treatment means were analyzed using one-way analysis of variance and separated using the least significant difference at 5% level of probability (P < 0.05).

Results

Cloning and protein motif analysis of KPvRxLR27 from Plasmopara viticola JN-9

KPvRxLR27 is one of 87 RxLR effectors identified in P. viticola JN-9 isolated from the Jeonju area of South Korea (unpublished data). Bioinformatics analysis using the aforementioned tools and blast analysis of KPvRxLR27 amino acid sequence against NCB database revealed that KPvRxLR27 highest amino acid identity with Chinese isolate (accession no. P0CV41.1) was 48.2%. KPvRxLR27 was therefore identified as a novel RxLR with protein sequence differing from previously published P. viticola RxLRs (Liu et al., 2018). KPvRxLR27 is a secreted effector with a full sequence open reading frame of 468 amino acids. It has an N-terminal signal peptide sequence, followed by an RxLR-EER motif, two nuclear localization signals (NLS1 and NLS2), and one N-glycosylation sites at the C-terminus (Fig. 1A and B).

Fig. 1

Protein alignment of KPvRxLR27 with three Plasmopara viticola RxLR effectors sequences. (A) Alignment of KPvRxL27 full-length amino acid sequence and three reported P. viticola RxLR effectors (RxLR70[P0CV21.1], RxLR101[P0CV39.1], and RxLR102[P0CV40.1]). The signal peptide sequence, RxLR, EER, N-glycosylation site, and two nuclear localizations signals motifs are marked with green, blue, yellow, red, and purple dotted lines respectively. (B) Structure and all functional protein motif of full-length KPvRxLR27 effector identified in the P. viticola JN-9 isolate. SP, signal peptide sequence; RxLR, arginine–any amino acid–leucine–arginine; NLS1, nuclear localization signal1; N186, N-glycosylation site at 186th amino acid; NLS2, nuclear localization signal 2.

P. viticola expresses KPvRxLR27 gene during infection

To verify whether KPvRxLR27 expressed during infection by P. viticola, we inoculated leaf discs of susceptible (Cabernet sauvignon) and resistant (Moldova) grape cultivars and performed RT-PCR at 0, 6, 12, 24, 48, 72, and 96 hours post-inoculation (hpi). Four days after infection with P. viticola, more advanced sporulation lesions were observed on susceptible grapes, while necrotic spots of resistance response occurred on resistant grape leaf discs. Also, in the early stages of infection (24 hpi), KPvRxLR27 expression levels were significantly higher in Cabernet sauvignon than in Moldova. Moreover, KPvRxLR27 expression was delayed in Moldova, with maximum expression achieved at the late stages of infection (96 hpi). In contrast, KPvRxLR27 effector achieved maximum expression at 72 hpi in Cabernet sauvignon (Fig. 2). These results show that KPvRxLR27 is a virulence factor, with gene expression occurring earlier and at higher levels in susceptible grapes compared to resistant ones upon pathogen infection.

Fig. 2

KPvRxLR27 expression in infected susceptible and resistant grape leaves. The image shows results of reverse transcription polymerase chain reaction depicting KPvRLR27 expression level in the susceptible (A) and resistant (C) grape cultivars at different times after P. viticola inoculation. (B, D) Graphical representation of the relative band intensity quantified from the agarose gel following KPvRxLR27 and reference genes expression in susceptible and resistant grape cultivars respectively.

KPvRxLR27 induces cell death in N. benthamiana and resistant grape cultivars

Plamoparara viticola RxLR effector plays two main important roles in modulating the host biochemical process to promote infection. To determine whether RxLR27 functions in promoting programmed cell death or repressing HR response, we transiently expressed KPvRxLR27 without its predicted signal peptide sequence by infiltrating transformed Agrobacterium into the leaves of N. benthamiana, susceptible (Cabernet sauvignon) and resistant (Moldova) grape cultivars. At 6 days’ post infiltration (dai), N. benthamiana leaf areas infiltrated with KPvRxLR27 exhibited visible cell death phenotypes corresponding to those induced by BAX and INF1, which were previously reported to trigger cell death in plants (Kamoun et al., 1998) (Fig. 3A). Measurement of electrolyte leakage in N. benthamiana infiltrated cells revealed that ion leakage at the site infiltrated with KPvRxLR27 was significantly (P < 0.05) higher than that at the site with the empty vector and which was as high as that observed in the positive controls (Fig. 3B). To investigate the resistance mechanisms underlying KPvRxLR27-induced cell death in N. benthamiana, we assessed the production of H2O2 at infiltration sites via DAB staining. The sites infiltrated by KPvRxLR27 exhibited dark brown foci at 3 days after infiltration, and the relative level of staining in KPvRxLR27-expressing leaves was high and similar to that in the BAX- and INF1-expressing leaves. On the other hand, the sites expressing the empty vector did not show any H2O2 accumulation (Fig. 3C). These findings suggest that KPvRxLR27 expression induces ROS accumulation leading to an oxidative burst, resulting in cell death as an immune response.

Fig. 3

KPvRxLR27 induces programmed cell death phenotype in Nicotiana benthamiana and resistant grapevine leaves. (A) KPvRxLR27 induces cell death in N. benthamiana. (B) Measurement of ion leakage to quantify cell death at the infiltrated sites. (C) 3,3′-diaminobenzidine (DAB) staining results showing the accumulation of H2O2 (dark grayish patches) in KPvRxLR27 expressing N. benthamiana leaves. (D) Cell death phenotypes after infiltration of empty vector and KPvRxLR27 into susceptible and resistant grape cultivars. Images of N. benthamiana and grapevine were captured at 7 and 4 days after infiltration (dai), respectively. All experiments were performed in triplicate (P < 0.05). The data show the mean expression ± standard deviation, *P < 0.05. BAX, Bcl-2-associated X; INF1, inverted fomin1.

To further confirm cell death-inducing ability of KPvRxLR27 effector, Agrobacterium carrying the pGR106–KPvRxLR27 recombinant was infiltrated into the third to sixth leaves of grape cultivars. KPvRxLR27 induced very rapid and obvious cell death (24–48 h after infiltration) Moldova (resistant grape cultivar) leaves, but not in the Cabernet sauvignon leaves (susceptible grape cultivar) (Fig. 3D). This is in line with the reported resistance reaction (necrotic spots) on the resistant cultivar leaf discs inoculated with P. viticola sporangia suspension (Semunyana et al., 2023). These results suggest that KPvRxLR27 induces cell death in non-host N. benthamiana and is recognized by host R-gene receptors to activate immune responses leading to cell death in resistant Vitis cultivars.

Subcellular localization of KPvRxLR27 in plant

In silico analysis (LOCALIZER motif scan) revealed that KPvRxLR27 contained two NLSs (NLS1 and NLS2) at its C-terminus. To determine the subcellular localization of this effector, GFP-tagged pCAMBIA1302 was fused to mature KPvRxLR27 and transiently expressed in N. benthamiana via agroinfiltration. Live cell imaging revealed that KPvRxLR27 produced fluorescent signals in the plasma membrane and nucleus (Fig. 4A). To determine whether nuclear localization is necessary for KPvRxLR27 cell death-inducing activity, we generated one mutant by deleting NLS1 motif (Fig. 4B) and agroinfiltrated it in N. benthamiana leaves. Indeed, none of the 12 leaf sites infiltrated by the mutant exhibited cell death whereas that infiltrated by the wild-type showed clear cell death at 6 dai (Fig. 4C). Additionally, NLS1 deletion mutant maintained nuclear location as the wild-type (Fig. 4A). These data suggest that KPvRxLR27 does not necessarily require nuclear localization to induce cell death in N. benthamiana.

Fig. 4

Subcellular localization of putative KPvRxLR27 and its mutants in plant. (A) Empty vector-GFP, KPvRxLR27 and its mutants fused with GFP were transiently expressed in Nicotiana benthamiana, and fluorescent signals were analyzed at 2 days after infiltration (dai) using a confocal microscope. (B) Schematic diagram of KPvRxLR27 wild-type and nuclear localization signal (NLS1) deletion mutant. (C) Functional analysis of the KPvRxLR27-predicted NLS1: the right side was infiltrated with KPvRxLR27 (WT), the left side with the empty vector, and sites 1–10 were infiltrated with NLS1 mutants.

Functional characterization of the putative N-glycosylation site

Similar to other secreted proteins, effectors undergo asparagine-mediated glycosylation for transport to the apoplast or inside the host cell. N-linked glycosylation (ALG3) of the effector LysM1 protein secreted by Magnaporthe oryzae was reported to be crucial for both the structure and function necessary for the pathogen to evade the host’s innate immune systems (Chen et al., 2014). Using Scan Prosite (https://prosite.expasy.org/scanprosite/), KPvRxLR27 was predicted to contain a single N-glycosylation site at Asn-186 (NFSG) of the C-terminus. To determine whether this N-glycosylation site plays an important role in KPvRxLR27 cell death-inducing activity, we induced an Asparagine-to-Alanine site-directed mutation (Fig. 5A) and transiently expressed the resulting KPvRxLR27N186A mutant in N. benthamiana leaves. Six days after infiltration, wild-type strain induced cell death in the leaves. However, the KPvRxLR27N186A mutant failed to induce cell death. Ion leakage measured to quantify cell death, was significantly lower at the site infiltrated by the mutant than the sites infiltrated by the wild-type (Fig. 5B and C). Moreover, the location of KPvRxLR27N186A was verified by confocal microscopy. Similar to the wild-type, KPvRxLR27N186A produced a fluorescence signal in both nucleus and cytosol (Fig. 4A). These results suggest that KPvRxLR27 is the most important post-translational modification for its cell death-inducing activity.

Fig. 5

Functional characterization of the KPvRxLR27-predicted N-glycosylation site. (A) Schematic diagram showing the asparagine-to-alanine site-directed mutation created at the KPvRxLR27 N-glycosylation site marked with red rectangles. (B) Transient expression of KPvRxLR27(wt) and its N-glycosylated mutant in Nicotiana benthamiana leaves. (C) Cell death quantified by measuring electrolyte leakage at infiltrated sites. Images of the hypersensitive response phenotype in N. benthamiana captured at 7 days after infiltration (dai). The least significant difference was determined at 5% probability (P < 0.05). The data show the mean expression ± standard deviation, **P < 0.01. BAX, Bcl-2-associated X; INF1, inverted fomin1.

Discussion

Effectors are protein molecules secreted by bacteria, nematodes, and oomycetes that modulate the plant immune system. During infection, P. viticola (a well-characterized oomycete) secretes effectors that govern the immune system of the host. Most reported effectors perform two main functions, whereby some act as suppressors, whereas others serve as inducers of host plant immunity (Arif et al., 2021; Brilli et al., 2018). Effectors induce immune response when they are recognized by the host receptors and become Avr, a phenomenon that is explained by gene-for-gene concept (Higgins et al., 1998).

In this study, functional analysis of KPvRxLR27, an RxLR effector from P. viticola JN-9 revealed that this effector induces cell death in both N. benthamiana and resistant grape cultivars. PvRxLR effectors have been reported to induce immune responses in the form of programmed cell death in N. benthamiana (Rothlin et al., 2021; Xiang et al., 2021). P. viticola effectors induce cell death in resistant grape; the conserved WY motif containing effectors but lacking proper RxLR motif induces necrosis in V. vinifera ‘Syrah (Combier et al., 2019). PvAvh77, a PvRxLR effector induces cell death in tobacco and Vtis riparia (a resistant grape cultivar) but not in Thompson seedless (a susceptible grape cultivar) (Fu et al., 2023). In this study, KPvRxLR27 induced cell death in N. benthamiana and resistant grape cultivar (Moldova) but, not in susceptible cultivar (Cabernet sauvignon) because resistant cultivar encodes genes for resistance R-proteins acquired during long-term co-evolution with P. viticola and other pathogens, facilitating the specific recognition of pathogen Avr proteins and induction of HR to limit infection (Eitas and Dangl, 2010). Moldova is a South Korean locally grown grape cultivars with reported resistance to downy mildew disease (Semunyana et al., 2023). However, the specific R-genes necessary for the recognition of KPvRxLR27 effector remains unknown, warranting further investigation.

Various effectors perform their functions in specific cell compartments, but nuclear localization is the most important target for most effectors that is necessary for their cell death-inducing activity (Chen et al., 2020; Liu et al., 2018). For instance, 66% of Hyaloperonospora arabidopsis effector candidates target either the nuclear region specifically or both nucleus and cytosol of the host cell (Caillaud et al., 2012). AVR1 nuclear localization is necessary to activate R-gene-mediated resistance (R1) (Du et al., 2015). Here, live cell imaging revealed that KPvRxLR27 exhibited fluorescence signals in the nucleus and plasma membrane, deletion of NLS1 impaired its cell death-inducing capacity. This may be because plant pathogenic microorganisms direct their effectors to the nucleus to modulate the host biological functions. Expression patterns detected by RT-PCR after inoculating the resistant and susceptible grape cultivars revealed that KPvRxLR27 achieved maximum expression at the early stages of infection in susceptible cultivar but showed very limited and delayed expression in the resistant cultivar. Early and high expression of PvRxLR5 and PvRxLR28 have been reported in P. viticola infected Thompson seedless (Xiang et al., 2016).

ETI is associated with plant resistance to pathogen infection resulting from the activation and increased expression of defense-related genes, accumulation of ROS, and flux of ions (Cui et al., 2015). Using DAB staining KPvRxLR27overexpression resulted in H2O2 accumulation in N. benthamiana leaves 72 h after infiltration, similar to a previous report that PvRxLR16 overexpression induces ROS accumulation and activation of other tobacco plant defense-related genes (Yin et al., 2019).

Effectors of filamentous fungi undergo N-glycosylation as an important post-translational modification to maintain structure stability and pathogenic function (Chen et al., 2014). Two N-glycosylation sites (PvAvh74N22 and PvAvh74N465) sites are important for effector cell death induction in P. viticola (Xiang et al., 2017). Similarly, KPvRxLR27 N-glycosylation site; N186(NSFG) was important for the effector cell death-inducing activity as transient expression of KPvRxLR27N184A mutant did not induce cell death in N. benthamiana which was observed in the wild-type.

This study revealed that the P. viticola secreted the RxLR effector KPvRxLR27 induces programmed cell death in N-glycosylation-dependent manner in the non-host, N. benthamiana. Additionally, KPvRxLR27 was recognized by the resistance R-gene when expressed in resistant grape cultivar. Therefore, KPvRxLR27 may function as an P. viticola JN-9 Avr gene, with its cell death-inducing activity depending on ETI as a defense response. However, further studies are necessary to confirm the identity of Vitis R-genes and elucidate the mechanisms underlying their interactions with Avr genes. To our knowledge, this study is the first report to demonstrate the functions of PvRxLR effector in South Korea, thereby contributing to the improvement of grape cultivar resistance to downy mildew disease.

Notes

Conflicts of Interest

No potential conflict of interest relevant to this work was reported.

Acknowledgments

This work was supported by the project No. PJ01492503 (CNU, Sang-Keun Oh) of Rural Development Administration, Republic of Korea.

Electronic Supplementary Material

Supplementary materials are available at The Plant Pathology Journal website (http://www.ppjonline.org/).

References

Arif S., Lim G. T., Kim S. H., Oh S.-K.. 2021;Characterization of Phytophthora capsici effector genes and their functional repertoire. Korean J. Agric. Sci. 48:643–654.
Bozkurt T. O., Schornack S., Banfield M. J., Kamoun S.. 2012;Oomycetes, effectors, and all that jazz. Curr. Opin. Plant Biol. 15:483–492.
Brilli M., Asquini E., Moser M., Bianchedi P. L., Perazzolli M., Si-Ammour A.. 2018;A multi-omics study of the grapevine-downy mildew (Plasmopara viticola) pathosystem unveils a complex protein coding- and noncoding-based arms race during infection. Sci. Rep. 8:757.
Caillaud M.-C., Piquerez S. J. M., Fabro G., Steinbrenner J., Ishaque N., Beynon J., Jones J. D. G.. 2012;Subcellular localization of the Hpa RxLR effector repertoire identifies a tonoplast-associated protein HaRxL17 that confers enhanced plant susceptibility. Plant J. 69:252–265.
Chen T., Liu R., Dou M., Li M., Li M., Yin X., Liu G.-T., Wang Y., Xu Y.. 2020;Insight into function and subcellular localization of Plasmopara viticola putative RxLR effectors. Front. Microbiol. 11:692.
Chen X.-L., Shi T., Yang J., Shi W., Gao X., Chen D., Xu X., Xu J.-R., Talbot N. J., Peng Y.-L.. 2014;N-glycosylation of effector proteins by an alpha-1,3-mannosyltransferase is required for the rice blast fungus to evade host innate immunity. Plant Cell 26:1360–1376.
Chisholm S. T., Coaker G., Day B., Staskawicz B. J.. 2006;Host-microbe interactions: shaping the evolution of the plant immune response. Cell 124:803–814.
Combier M., Evangelisti E., Piron M.-C., Rengel D., Legrand L., Shenhav L., Bouchez O., Schornack S., Mestre P.. 2019;A secreted WY-domain-containing protein present in European isolates of the oomycete Plasmopara viticola induces cell death in grapevine and tobacco species. PLoS ONE 14:e0220184.
Cui H., Tsuda K., Parker J. E.. 2015;Effector-triggered immunity: from pathogen perception to robust defense. Annu. Rev. Plant Biol. 66:487–511.
Dodds P. N., Rathjen J. P.. 2010;Plant immunity: towards an integrated view of plant-pathogen interactions. Nat. Rev. Genet. 11:539–548.
Du Y., Berg J., Govers F., Bouwmeester K.. 2015;Immune activation mediated by the late blight resistance protein R1 requires nuclear localization of R1 and the effector AVR1. New Phytol. 207:735–747.
Eitas T. K., Dangl J. L.. 2010;NB-LRR proteins: pairs, pieces, perception, partners, and pathways. Curr. Opin. Plant Biol. 13:472–477.
Fu Q., Wang Y., Yang J., Jiao Y., Li W., Yang F., Yin X., Shang B., Liu R., Zhang Y., Saileke A., Liu G., van Nocker S., Hu H., Xu Y.. 2023; Plasmopara viticola RxLR effector PvAvh77 triggers cell death and governs immunity responses in grapevine. J. Exp. Bot. 74:2047–2066.
Higgins V. J., Lu H., Xing T., Gelli A., Blumwald E.. 1998;The gene-for-gene concept and beyond: interactions and signals. Can. J. Plant Pathol. 20:150–157.
Jones J. D. G., Dangl J. L.. 2006;The plant immune system. Nature 444:323–329.
Kamoun S.. 2006;A catalogue of the effector secretome of plant pathogenic oomycetes. Annu. Rev. Phytopathol. 44:41–60.
Kamoun S., van West P., Vleeshouwers V. G. A. A., de Groot K. E., Govers F.. 1998;Resistance of Nicotiana benthamiana to Phytophthora infestans is mediated by the recognition of the elicitor protein INF1. Plant Cell 10:1413–1425.
Lacomme C., Santa Cruz S.. 1999;Bax-induced cell death in tobacco is similar to the hypersensitive response. Proc. Natl. Acad. Sci. U. S. A. 96:7956–7961.
Lee S. B., Milgroom M. G., Taylor J. W.. 1988;A rapid, high yield mini-prep method for isolation of total genomic DNA from fungi. Fungal Genet. Rep. 35:11.
Liu Y., Lan X., Song S., Yin L., Dry I. B., Qu J., Xiang J., Lu J.. 2018; In planta functional analysis and subcellular localization of the oomycete pathogen Plasmopara viticola candidate RXLR effector repertoire. Front. Plant Sci. 9:286.
Ngou B. P. M., Ahn H.-K., Ding P., Jones J. D. G.. 2021;Mutual potentiation of plant immunity by cell-surface and intracellular receptors. Nature 592:110–115.
Oh S.-K., Young C., Lee M., Oliva R., Bozkurt T. O., Cano L. M., Win J., Bos J. I. B., Liu H.-Y., van Damme M., Morgan W., Choi D., Van der Vossen E. A. G., Vleeshouwers V. G. A. A., Kamoun S.. 2009; In planta expression screens of Phytophthora infestans RXLR effectors reveal diverse phenotypes, including activation of the Solanum bulbocastanum disease resistance protein Rpi-blb2. Plant Cell 21:2928–2947.
Pruitt R. N., Locci F., Wanke F., Zhang L., Saile S. C., Joe A., Karelina D., Hua C., Fröhlich K., Wan W.-L., Hu M., Rao S., Stolze S. C., Harzen A., Gust A. A., Harter K., Joosten M. H. A. J., Thomma B. P. H. J., Zhou J.-M., Dangl J. L., Weigel D., Nakagami H., Oecking C., Kasmi F. E., Parker J. E., Nürnberger T.. 2021;The EDS1-PAD4-ADR1 node mediates Arabidopsis pattern-triggered immunity. Nature 598:495–499.
Rothlin C. V., Hille T. D., Ghosh S.. 2021;Determining the effector response to cell death. Nat. Rev. Immunol. 21:292–304.
Semunyana M., Kim S. H., Min J., Lee S.-M., Oh S.-K.. 2023;Identification of host-resistant and susceptible varieties of Korean grapes to Plasmopara viticola, a pathogen causing grapevine downy mildew. Korean J. Mycol. 51:179–190.
Xiang G., Yin X., Niu W., Chen T., Liu R., Shang B., Fu Q., Liu G., Ma H., Xu Y.. 2021;Characterization of CRN-like genes from Plasmopara viticola: searching for the most virulent ones. Front. Microbiol. 12:632047.
Xiang J., Li X., Wu J., Yin L., Zhang Y., Lu J.. 2016;Studying the mechanism of Plasmopara viticola RxLR effectors on suppressing plant immunity. Front Microbiol. 7:709.
Xiang J., Li X., Yin L., Liu Y., Zhang Y., Qu J., Lu J.. 2017;A candidate RxLR effector from Plasmopara viticola can elicit immune responses in Nicotiana benthamiana. BMC Plant Biol. 17:75.
Yin L., An Y., Qu J., Li X., Zhang Y., Dry I., Wu H., Lu J.. 2017;Genome sequence of Plasmopara viticola and insight into the pathogenic mechanism. Sci. Rep. 7:46553.
Yin X., Shang B., Dou M., Liu R., Chen T., Xiang G., Li Y., Liu G., Xu Y.. 2019;The nuclear-localized RxLR effector PvAvh74 from Plasmopara viticola induces cell death and immunity responses in Nicotiana benthamiana. Front. Microbiol. 10:1531.

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Fig. 1

Protein alignment of KPvRxLR27 with three Plasmopara viticola RxLR effectors sequences. (A) Alignment of KPvRxL27 full-length amino acid sequence and three reported P. viticola RxLR effectors (RxLR70[P0CV21.1], RxLR101[P0CV39.1], and RxLR102[P0CV40.1]). The signal peptide sequence, RxLR, EER, N-glycosylation site, and two nuclear localizations signals motifs are marked with green, blue, yellow, red, and purple dotted lines respectively. (B) Structure and all functional protein motif of full-length KPvRxLR27 effector identified in the P. viticola JN-9 isolate. SP, signal peptide sequence; RxLR, arginine–any amino acid–leucine–arginine; NLS1, nuclear localization signal1; N186, N-glycosylation site at 186th amino acid; NLS2, nuclear localization signal 2.

Fig. 2

KPvRxLR27 expression in infected susceptible and resistant grape leaves. The image shows results of reverse transcription polymerase chain reaction depicting KPvRLR27 expression level in the susceptible (A) and resistant (C) grape cultivars at different times after P. viticola inoculation. (B, D) Graphical representation of the relative band intensity quantified from the agarose gel following KPvRxLR27 and reference genes expression in susceptible and resistant grape cultivars respectively.

Fig. 3

KPvRxLR27 induces programmed cell death phenotype in Nicotiana benthamiana and resistant grapevine leaves. (A) KPvRxLR27 induces cell death in N. benthamiana. (B) Measurement of ion leakage to quantify cell death at the infiltrated sites. (C) 3,3′-diaminobenzidine (DAB) staining results showing the accumulation of H2O2 (dark grayish patches) in KPvRxLR27 expressing N. benthamiana leaves. (D) Cell death phenotypes after infiltration of empty vector and KPvRxLR27 into susceptible and resistant grape cultivars. Images of N. benthamiana and grapevine were captured at 7 and 4 days after infiltration (dai), respectively. All experiments were performed in triplicate (P < 0.05). The data show the mean expression ± standard deviation, *P < 0.05. BAX, Bcl-2-associated X; INF1, inverted fomin1.

Fig. 4

Subcellular localization of putative KPvRxLR27 and its mutants in plant. (A) Empty vector-GFP, KPvRxLR27 and its mutants fused with GFP were transiently expressed in Nicotiana benthamiana, and fluorescent signals were analyzed at 2 days after infiltration (dai) using a confocal microscope. (B) Schematic diagram of KPvRxLR27 wild-type and nuclear localization signal (NLS1) deletion mutant. (C) Functional analysis of the KPvRxLR27-predicted NLS1: the right side was infiltrated with KPvRxLR27 (WT), the left side with the empty vector, and sites 1–10 were infiltrated with NLS1 mutants.

Fig. 5

Functional characterization of the KPvRxLR27-predicted N-glycosylation site. (A) Schematic diagram showing the asparagine-to-alanine site-directed mutation created at the KPvRxLR27 N-glycosylation site marked with red rectangles. (B) Transient expression of KPvRxLR27(wt) and its N-glycosylated mutant in Nicotiana benthamiana leaves. (C) Cell death quantified by measuring electrolyte leakage at infiltrated sites. Images of the hypersensitive response phenotype in N. benthamiana captured at 7 days after infiltration (dai). The least significant difference was determined at 5% probability (P < 0.05). The data show the mean expression ± standard deviation, **P < 0.01. BAX, Bcl-2-associated X; INF1, inverted fomin1.