Escherichia coli Top10 used for DNA cloning was selectively cultured in solid or liquid Luria-Bertani (LB) media containing kanamycin (50 mg/L) at 37°C. Agrobacterium tumefaciens GV3101 was cultured at 28°C in solid or liquid LB media containing kanamycin (50 mg/L), gentamycin (30 mg/L), and rifampicin (50 mg/L). Transformations of DNA into Top10 and GV3101 were conducted following standard protocols for heat shock treatment and freezing-thawing transformation, respectively.

Plasmopara viticola YL was preserved in the lab by inoculating a sporangium suspension of the strain onto young leaves of V. vinifera Thompson Seedless every 5–7 days. Phytophthora capsici was maintained by subculturing the mycelia on V8 juice medium (2.5% V8, 1.5% agar) at 25°C in the dark.

In vitro grown plantlets of grapevine V. riparia and V. vinifera Thompson Seedless, which were intended for use in the agrobacterium-mediated gene transient expression experiments, were primarily cultured in jars with medium containing half-strength Murashige and Skoog medium (MS), 30 g/L sucrose, 0.1 mg/L IBA (indol-3-butyric acid), 0.3 mg/L 6-BA (6-benzylaminopurine), and 3 g/L phytagel. The in vitro plants were propagated in the medium with half-strength MS, 15 g/L sucrose, 0.15 mg/L IBA, and 3 g/L phytagel. The environmental temperature of the tissue culture system was 24 ± 2°C, and the photoperiod was set to 16 h illumination: 8 h dark. Plants of V. vinifera Pinot Noir were grown in the Grape Germplasm Resources Repository at Northwest A&F University, Yangling, China. Detached grapevine leaves inoculated with P. viticola YL were incubated in a climate chamber with same culture conditions as the in vitro plants. Nicotiana benthamiana seedlings were grown in pots with a matrix composed of peat and vermiculite at 22 ± 2°C under a 16 h photoperiod and 75% relative humidity in a greenhouse.

The protein sequences encoded by the CRN genes cloned from the P. viticola YL genome were translated using the ExPASy-Translate tool¹. Then, protein physical and chemical parameters were predicted using ExPASy-ProtParam². Signal peptide prediction of PvCRN proteins was performed using the SignalP 3.0 server³ and Phobius⁴. For prediction with SignalP 3.0, both neural networks (NN) and hidden Markov models (HMM) methods were used. The prediction criteria were as follows: the sequence was labeled “yes” when the S-score was greater than the default threshold 0.47 under SignalP-NN prediction, and for SignalP-HMM prediction, the sequence was labeled “yes” when the S-probability was greater than the default threshold 0.5 (Sperschneider et al., 2015). The presence of signal peptides was finally determined by combining the prediction results of the two software programs. The presence of importin α-dependent nuclear localization signal (NLS) peptides was predicted by the NLS Mapper server⁵. Transmembrane helices were predicted using Predictprotein⁶ and TMpred server⁷. Multiple sequence alignment of PvCRN proteins was conducted in ClustalX-2.1, and the results were converted into colored images using CLC Sequence Viewer 8.0. The most conserved N-terminal motifs among the PvCRN proteins were identified, and the corresponding sequence logos were generated by Weblogo⁸. The presence of the effector subdomains defined in Phytophthora species (Haas et al., 2009; Stam et al., 2013) in the C-terminal region of PvCRN proteins were identified by multiple sequence alignment with CLC Sequence Viewer 8.0. Phylogenetic analysis was performed using MEGA7.0, using the maximum likelihood method with the best-fit substitution model WAG + G (Zuckerkandl and Pauling, 1965; Kumar et al., 2016). Phylogenetic trees for display were drawn with the iTOL server⁹. Conserved functional domains and motifs of PvCRN proteins were predicted using the online software Pfam 33.1¹⁰, SMART¹¹, and ExPASy-ScanProsite¹².

A sporangium suspension of 50,000 sporangia/mL of P. viticola YL was sprayed on detached leaves of V. vinifera Pinot Noir, which had been sterilized with 1% sodium hypochlorite, for propagation (Mestre et al., 2012). After the sporangiophores of P. viticola YL reached out from the abaxial surface of grapevine leaves, the sporangia were collected into sterilized water using a soft brush, centrifuged, and stored at –80°C. Genomic DNA was extracted from P. viticola YL using the CTAB method (Xin and Chen, 2012). For gene expression analysis, P. viticola YL sporangia were inoculated onto detached Pinot Noir leaves in the same manner described above. Different batches of inoculated grapevine leaves were collected at 0, 24, 48, 72, 96, and 120 h post inoculation. The whole experiment was repeated three times. All samples collected were stored at –80°C before use. The hybrid total RNA of grapevine leaves and P. viticola YL mycelia was collected at different time points and isolated using the E.Z.N.A.^® Plant RNA Kit (Omega Bio-tek, United States).

The coding sequences of the PvCRN genes, excluding the fragments that were predicted with high probability to be related to signal peptides, were amplified from the genomic DNA of P. viticola YL using a KOD-Plus-Neo DNA Polymerase Kit (TOYOBO, Japan) with specific primer pairs (Supplementary Table 1). The DNA sequences predicted to encode signal peptides were cloned using the same method. PvCRN genes without signal peptide coding sequences were inserted into the plant virus-based expression binary vector PVX1 that was modified from the pGR106/107 vector with two restriction enzyme sites (SmaI and Not I). After confirmation by sequencing, the corresponding DNA was subcloned into the plant expression vector pCAMBIA2300-GFP (Su et al., 2018). The signal peptide coding sequences were introduced to the signal peptide trap vector pSUC2 (pSUC2T7M13ORI) and confirmed by sequencing. The first-strand cDNA was synthesized using a PrimeScript^TM II 1st Strand cDNA Synthesis Kit (Takara, Japan) with mixed total RNA (2 μg) as the template. Then, the cDNA was diluted with an equal volume of nuclease-free water. Expression of each PvCRN gene at different time points during the development of downy mildew was detected by performing reverse transcription-PCR (RT-PCR) on the diluted cDNA templates (Liu et al., 2018). The PCR procedure was as follows: a pre-denaturing step at 94°C for 3 min, a 33-cycle reaction consisting of denaturation at 94°C for 30 s, annealing at 54°C for 30 s, and extension at 72°C for 2 min, followed by a final elongation at 72°C for 8 min. The PCR products were separated by 1% agarose gel electrophoresis and detected on a gel imaging system.

The plasmid constructs harboring PVX1-PvCRN and pCAMBIA2300-PvCRN-GFP were transformed into Agrobacterium tumefaciens strain GV3101 using the freezing-thawing method. After confirmation by selective culture and PCR testing, the transformants were propagated in a shaking culture in LB liquid media with appropriate antibiotics at 28°C. The bacterial cells were collected by centrifugation (5000 rpm × 3 min), washed twice with 10 mM MgCl₂ solution, and resuspended in the infiltration buffer (10 mM MES, pH 5.7, 10 mM MgCl₂, and 200 μM acetosyringone), followed by incubation at 28°C for 3 h before infiltration. The bacterial suspension was diluted to an OD₆₀₀ of 0.4 with the infiltration buffer for each plasmid construct and then injected into N. benthamiana leaves from the abaxial surface with a 1 mL syringe without a needle. For cell death suppression assays, the left and right sides of the N. benthamiana leaves were agroinfiltrated by A. tumefaciens containing either GFP or PvCRN genes, respectively, and the infiltrated areas were marked. Twelve h after the first infiltration, the marked areas of the leaves were re-infiltrated by A. tumefaciens carrying either the PVX-INF1 or PVX-Bax constructs. For subcellular localization analysis of the PvCRN proteins, the OD₆₀₀ of the agrobacteria transformed with the pCAMBIA2300-PvCRN-GFP construct was adjusted to 0.3. For measurements of the transient expression of each PvCRN gene in Vitis spp., A. tumefaciens cells were resuspended in grapevine infiltration buffer (10 mM MES, pH 5.7, 10 mM MgCl₂, and 500 μM acetosyringone), and the OD₆₀₀ for each plasmid construct was adjusted to 1.0. The bacteria were injected into the top right and bottom left parts of the abaxial surfaces of the in vitro grapevine leaves with a needleless 1 mL syringe (Zottini et al., 2008). A. tumefaciens cells containing pCAMBIA2300-GFP or pCAMBIA2300-RXLR77-GFP were injected into the lower right and upper left parts of the abaxial surface, respectively, of the same leaves expressing each PvCRN to serve as controls. The infiltrated grapevine leaves were monitored for the expression of genes of interest for 10 days post infiltration, and the phenotypes of the leaves expressing genes of interest were recorded.

The subcellular localization of the PvCRN proteins in plant cell were determined by observing the fluorescence of transiently expressed PvCRN-GFP proteins in N. benthamiana leaves 48–72 h after agroinfiltration with confocal microscopy, following the standard protocol. The nuclear localization of the proteins was marked by co-expression of the construct pYJ-NLS-mCherry with PvCRN-GFP proteins. The plasma membrane-localized marker PM-RK (Zhu et al., 2014) was co-expressed with each PvCRN protein to validate the plasma membrane localization of proteins. For each transiently expressed PvCRN-GFP protein that had been introduced to in vitro grapevine leaves by means of agroinfiltration, expression was validated by observing the GFP fluorescence of the recombinant protein using fluorescence microscopy, as described in a previous study (Zottini et al., 2008).

The secretion function of the putative signal peptide sequences in the PvCRN proteins was verified using a previously described yeast signal sequence trap assay (Oh et al., 2009). Briefly, plasmid constructs of the putative signal peptide coding sequence (pSUC2-PvCRNXSP) were transformed into the invertase-negative yeast strain YTK12 using the lithium acetate method, and the transformants were obtained by selective culture on CMD-W medium (0.67% yeast nitrogen base without amino acids, 0.075% minus Trp dropout Supplement, 2% sucrose, 0.1% glucose, and 2% agar). The positive clones were then transferred to YPRAA medium (1% yeast extract, 2% peptone, 2% raffinose, and 2% agar) to verify invertase secretion. The invertase activity was determined by monitoring the reduction of 2,3,5-triphenyl tetrazolium chloride (TTC) to the insoluble, red-colored triphenylformazan. Signal peptide sequences of the effectors Avr1b and Mg87 were included in the experiment as positive and negative controls, respectively.

Fresh P. capsici mycelia were cut into small pieces and cultured in liquid V8 medium for approximately 4 days to produce sporangia. Then, the mycelia containing sporangia were washed twice with cold sterilized water (4°C) and incubated at 4°C in water to induce zoospore release. The concentration of zoospores was measured with a hemocytometer, and the final zoospore concentration used in inoculations was adjusted to 50 zoospores/μL. N. benthamiana leaves that had been infiltrated with agrobacteria containing the indicated plasmid constructs were detached at 48 h post infiltration (hpi), inoculated with 10 μL droplets of P. capsici zoospores on their abaxial surfaces, and then incubated in plastic trays with sufficient moisture at 25°C for 48 h. The symptoms and lesion lengths for all lesions were recorded for 9–12 infected leaves, and the experiment was repeated at least three times (Song et al., 2015). Statistical analysis was conducted in Excel and GraphPad Prism 6.

Nicotiana benthamiana leaves infiltrated with agrobacteria carrying the genes of interest were collected at 48–72 hpi, immediately frozen in liquid nitrogen, and stored at –80°C before western blot analysis. After being ground to a powder, the samples were transferred into microtubes with protein extraction buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 10% glycerol, 0.2% Triton X-100, 0.1% NP-40, 0.25% sodium deoxycholate, 2 mM ethylenediaminetetraacetic acid [pH 8.0], 5 mM dithiothreitol, and 2 mM phenylmethylsulphonyl fluoride) and gently vortexed intermittently for 40 min. The samples were then centrifuged at room temperature (12000 rpm × 10 min) to collect the supernatants. The supernatants were boiled with 5 × SDS-loading buffer and loaded into 12% SDS-PAGE gels for separation. Gels were blotted onto a PVDF membrane (Merck, Germany) with a semi-dry gel transfer instrument (Bio-Rad, United States), followed by blocking the membrane in 5% skimmed milk dissolved in TBST (20 mM Tris-HCl [pH 8.0], 150 mM NaCl, and 0.05% (v/v) Tween 20). Membranes were incubated with the primary antibody mouse anti HA-Tag mAb or anti-GFP mAb (ABclonal) at a ratio of 1: 3000 at 4°C overnight. After washing three times with TBST, the membrane was incubated with the corresponding horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (H + L) secondary antibody (ABclonal) at a ratio of 1:5000 at room temperature for 2 h. Finally, the target proteins were detected by enhanced chemiluminescence (Beyotime, China) after the membrane was washed four times with TBST.

In P. viticola isolate YL, 35 functional CRN-like genes were predicted from its genome sequence data¹³, and 27 out of 35 CRN-like gene coding sequences (PvCRN) were successfully cloned and verified. The NCBI accession number and the DNA sequence associated with each PvCRN gene are provided in Supplementary Table 2. All cloned PvCRN sequences were aligned with the genome sequences of other P. viticola isolates, including those of JL-7-2, INRA-PV221, and PVitFEM01 (Dussert et al., 2016; Yin X. et al., 2017; Brilli et al., 2018) using the NCBI-BLAST program. All the PvCRN genes in YL shared high similarities with the CRN-like DNA sequences of the other three P. viticola isolates (data not shown), but a few CRN-like genes showed 100% identity with the cognate DNA sequences from the other three genomes (Table 1). The molecular weights of the PvCRN proteins ranged from 16.51 to 84.22 kDa, with most members having molecular weights below 50 kDa (Table 2). All PvCRN proteins have the conserved motifs L (/V) -X-L-Y (/F)-L-A-K (/R/H) and I-H-V-L-V-X-X-P present at their N-terminal regions (Supplementary Figures 1, 2). In addition, two conserved motifs, V-X-L-X-C-A-X-V (/Y)-G and W-L (/M) were also found at the N-terminal regions of the PvCRN proteins (Supplementary Figures 1, 2). In contrast, the C-terminal sequences of the PvCRN family proteins were diverse, and no new CRN-related motif was found (Supplementary Figure 2). Only a few C-terminal subdomains reported in Phytophthora spp. were matched to a few PvCRN proteins (Table 2).

There are eight PvCRN proteins predicted to have classical signal peptide sequences, whereas most PvCRN proteins showed no classical secretion features (Table 2). Most PvCRN proteins (21 out of 27) were predicted to have classical NLS. Meanwhile, most PvCRN proteins (20 out of 27) seem to form transmembrane helix structures. Some PvCRN proteins also contain some conserved functional domains or motifs in their C-termini; for example, PvCRN31 contains the protein kinase domain (Prosite entry PS50011). The HNH_2 motif (PF13391), which is related to DNA binding, was found both in PvCRN16 and PvCRN23 in the C-terminal region (Table 2).

High sequence similarities were found within several groups of PvCRN genes, indicating that gene duplication has occurred during the evolution of PvCRN genes. PvCRN18, PvCRN27, and PvCRN29 probably developed from one ancestral gene (Supplementary Figure 3). In particular, PvCRN27 and PvCRN29 could be paralogs produced from gene tandem duplication as the distance between these two loci is less than 100 kb (data not shown). Similarly, PvCRN10 and PvCRN11 could also be paralogs developed from gene duplication (Supplementary Figure 4).

In addition, gene recombination was also found among PvCRN genes. Three types of gene recombination phenomena were significantly detected by RDP5. First, PvCRN genes shared a highly conserved N-terminal coding sequence but differed in their C-terminal coding sequences. Second, PvCRN genes shared a highly conserved C-terminal coding sequence but displayed diversity in the N-terminal coding sequence (Table 3). Third, one PvCRN gene was a recombinant gene composed of different fragments from at least two other PvCRN genes. For example, PvCRN15 is a recombinant gene of PvCRN26 and PvCRN23, with the former providing most of the 5’ terminal fragment and the entire 3′ terminal coding sequence, and the latter providing the remainder of the PvCRN15 N-terminal coding sequence. PvCRN16 is derived from the partial 5′ end sequence and the entire 3′ terminal sequence of PvCRN23, with the rest of the 5′ terminal sequence derived from PvCRN26. For PvCRN21, the major parent was PvCRN9, while the minor parent was predicted to be PvCRN31(Table 3 and Supplementary Table 3).

The mature protein sequences of all 27 PvCRNs (the predicted classical signal peptide sequences were excluded for the eight PvCRNs mentioned above) were aligned with ClustalX-2.1; the alignment is available in Supplementary Figure 5. The alignment was then submitted to MEGA 7.0 for phylogenetic analysis. A phylogenetic tree (Figure 1) with 50% bootstrap cutoffs showed that PvCRN proteins cloned from P. viticola YL were divided into seven clades, including five groups and two singletons, suggesting that PvCRN genes are actively evolving new features and functions despite retaining several conserved motifs. Compared to the CRN-like proteins of the sunflower downy mildew causal agent P. halstedii (hereafter referred to as PhCRN proteins, and the sequence information is available at https://www.ncbi.nlm.nih.gov/genome/?term=txid4781[Organism:noexp]), no PvCRN proteins clustered together with PhCRN proteins in a phylogenetic tree with 50% bootstrap cutoffs, indicating that the phylogenetic relationships between most PvCRN genes and PhCRN genes are distant (Figure 2). The complete alignment of CRN proteins from P. viticola YL and P. halstedii is available in Supplementary Figure 6.

Eight predicted signal peptide coding sequences of the PvCRN gene family were cloned into the yeast invertase vector pSUC2 and were subsequently transformed into the invertase-negative yeast strain YTK12. Yeast transformants expressing PvCRN signal peptides (PvCRN-SP), including PvCRN1-SP, PvCRN9-SP, PvCRN10-SP, and PvCRN23-SP, were able to grow rapidly on CMD-W and YPRAA media; the positive control (Avr1b) was also able to grow well on these media. Compared to the positive control, the transformants expressing PvCRN2-SP, PvCRN7-SP, PvCRN21-SP, PvCRN22-SP, and the negative control exhibited negligible growth (Figure 3A). However, in the invertase activity assay, all the predicted signal peptides of the PvCRN proteins transported invertase from yeast transformants into the sucrose solution, leading to the color reaction (Figure 3B). Overall, at least four of the eight predicted PvCRN-SPs were validated to secrete proteins.

With GFP fused to the C-terminus of each PvCRN protein (excluding the signal peptide) expressed in N. benthamiana leaves, the subcellular localization of PvCRN proteins was determined by fluorescence microscopic imaging. The results showed that most PvCRN proteins (19 out of 27) were diffusely localized in the plasma membrane, cytoplasm, and nucleus of the plant cell (Figure 4A and Supplementary Figure 7). Moreover, four PvCRN proteins were mainly distributed in the plant cell plasma membrane, including PvCRN15, PvCRN16, PvCRN30, and PvCRN35. In addition, PvCRN15 and PvCRN35 were probably localized in both the plasma membrane and the nuclear envelope (Figure 4B). Only three PvCRN proteins (PvCRN19, PvCRN27, and PvCRN29) with high probabilities of NLS presence (Table 2) were specifically localized in the plant cell nucleus (Figure 4C). Particularly, PvCRN17 was mainly localized in the plasma membrane and nucleus of the plant cell (Figure 4D). To confirm the membrane and nuclear localization of the proteins, some PvCRN proteins were selected to verify their co-localization with the membrane protein PM-RK linked with mCherry or the PV40-NLS-guided mCherry protein, respectively. Most PvCRN proteins were localized in the plasma membrane and nucleus, in agreement with the presence of transmembrane helices and predicted NLS.

Pathogen effectors are recognized by host plant R proteins directly or indirectly during long-term co-evolution between plants and their pathogens. Upon recognition of the avirulent effector (Avr) by the plant NB-LRR protein (R), hypersensitive response (HR) is induced and usually coupled with local cell death in the host plant, preventing further biotrophic pathogen propagation (Petit-Houdenot and Fudal, 2017). Hence, it is important to identify plant disease resistance-related components or R genes to identify the cell death-inducing effectors of biotrophic pathogens. The North American Vitis species, V. riparia, is partially resistant to P. viticola, and most of its accessions show HR-related necrosis after infection by P. viticola (Cadle-Davidson, 2008; Gómez-Zeledón et al., 2013). The cell death-inducing activity of PvCRN proteins was tested on leaves of N. benthamiana and V. riparia with the aim of identifying some Avr-like effectors. As shown in Table 4 and Supplementary Figure 8, most PvCRN proteins (26 out of 27) did not induce plant cell death in N. benthamiana leaves, whereas INF1, the positive control protein, did induce cell death. No PvCRN proteins were able to induce veritable cell death in V. riparia leaves besides PvRXLR77 (Table 4 and Supplementary Figure 9), which is the ortholog of RXLR_PVITv1008311 in P. viticola isolate PvitFEM01 and reported to elicit HR-like cell death in V. riparia (Brilli et al., 2018). In particular, PvCRN11 induced spotted necrosis on N. benthamiana leaves but left no apparent phenotypic changes on V. riparia and V. vinifera cv. Thompson Seedless leaves (Figure 5). To confirm that PvCRN proteins were expressed in grapevine leaves, the GFP fluorescence of each PvCRN-GFP recombinant protein was verified prior to recording the phenotype of the infiltrated area (Supplementary Figure 10). Overall, the result of the test for cell death-inducing activity on V. riparia among the PvCRN proteins was in accordance with the result that most PvCRN proteins failed to induce necrosis on N. benthamiana. Even when overexpressed in grapevine leaves, no PvCRN protein induced apparent phenotypic changes in the host.

It is beneficial for plants to execute autonomous cell death to prevent the colonization of biotrophic and semi-biotrophic pathogens. For example, local necrosis or hypersensitive cell death is observed when PTI or ETI is triggered in a plant. Despite the fact that different plants are infected by different pathogens, the PTI responses in different plants share conserved molecular mechanisms (Boller and Felix, 2009; Faulkner and Robatzek, 2012; Bigeard et al., 2015), which are aimed at achieving similar outcomes. Programmed cell death (PCD) in plants is one of the manifestations of hypersensitive response and shares conserved hallmarks and some conserved regulators with PCD in animals (Reape and McCabe, 2008, 2010). For example, Bax, a proapoptotic protein from mice, activates PCD in some plant species, such as Nicotiana species and Arabidopsis thaliana (Lacomme and Santa Cruz, 1999; Kawai-Yamada et al., 2001; Yoshinaga et al., 2005). However, V. riparia leaves transiently overexpressing Bax-GFP recombinant protein simply turned brown on the abaxial side but did not show strong necrosis (Supplementary Figures 9, 10), probably because Bax-induced PCD in Vitis species depends on additional factors such as sufficient light (Yoshinaga et al., 2005) or biotrophic pathogen challenge. To overcome these plant defense responses and ensure sustainable access to nutrients, biotrophic pathogens secrete virulent effectors to inhibit cell death processes, keeping the host tissue alive.

It has been well documented that HR-like cell death is highly important for plant resistance to biotrophic pathogens (Glazebrook, 2005; Zebell and Dong, 2015). In accordance with that, biotrophs produce virulent effectors that deactivate host cell death responses (Chaudhari et al., 2014). To determine the virulence of the PvCRN proteins, each PvCRN gene was co-expressed with one of the cell death inducers: INF1or Bax. With INF1 expressed slightly later than each PvCRN protein in N. benthamiana leaves, the ability of PvCRN proteins to suppress PTI-related cell death was measured. Most PvCRN proteins failed to suppress cell death triggered by INF1 (INF1-CD); only PvCRN20 could completely suppress INF1-CD in N. benthamiana leaves (Table 4 and Supplementary Figure 11). In addition, PvCRN2, PvCRN16, and PvCRN17 partially suppressed or delayed INF1-CD (Supplementary Figure 11).

As for the suppressive effects of PvCRN proteins on Bax-induced plant cell death (Bax-CD), each PvCRN gene was delivered by A. tumefaciens at the same time as the Bax gene and 12 h before Bax. All PvCRN proteins failed to block Bax-CD when each of them was expressed simultaneously with Bax. However, when each PvCRN protein was able to accumulate earlier than Bax, some showed an inhibitory effect on Bax-CD. Altogether, 15 PvCRN proteins significantly suppressed plant Bax-induced PCD (Table 4 and Supplementary Figure 11). Overall, PvCRN20 fully prevented cell death induced by either INF1 or Bax in N. benthamiana leaves. Meanwhile, both PvCRN16 and PvCRN17 attenuated cell death induced by INF1or Bax in N. benthamiana leaves.

As 15 PvCRN proteins showed the ability to suppress plant cell death, thus contributing to plant resistance to (semi-) biotrophic pathogens, their effects on the susceptibility of N. benthamiana to the semi-biotrophic pathogen P. capsici were measured to further assess their virulence. Among the PvCRN proteins that suppressed Bax-CD, PvCRN17, PvCRN20, and PvCRN23 produced lesions with lengths that were significantly greater than those of the control GFP (Figure 6 and Table 5), thus promoting P. capsici colonization of N. benthamiana leaves. In contrast, PvCRN10 and PvCRN26 enhanced the resistance of N. benthamiana leaves to P. capsici (Figure 7 and Table 5). The other 10 PvCRN proteins showed no effect on the resistance of N. benthamiana leaves to P. capsici (Table 5 and Supplementary Figure 12). In addition, PvCRN1, a PvCRN protein that neither induced plant cell death nor suppressed INF1- or Bax-induced plant cell death, promoted resistance to P. capsici in N. benthamiana leaves. In addition, PvCRN31, which contains a protein kinase domain (Prosite entry PS50011) at its C-terminus, did not affect the susceptibility of N. benthamiana leaves to P. capsici (Supplementary Figure 13).

The nucleus-localized PvCRN proteins PvCRN19, PvCRN27, and PvCRN29 had different effects on disease resistance in N. benthamiana. PvCRN19 significantly enhanced the susceptibility of N. benthamiana leaves to P. capsici, whereas PvCRN27 and PvCRN29 improved the resistance level of N. benthamiana to the pathogen (Figure 8). This suggests that they differed in virulence, although these proteins were all translocated to the plant nucleus.

In addition, PvCRN11, which is the only one that induced necrosis in N. benthamiana, repressed the extension of P. capsici lesions on N. benthamiana leaves (Figure 9). This indicates that PvCRN11 is recognized by host plants and triggers host defense responses.

The transcription pattern of each PvCRN gene likely reflects the involvement of these genes in the interaction between P. viticola and the grapevine. The transcription levels of the PvCRN genes were detected by RT-PCR at 96 hpi, which was selected because the hyphae and haustoria of P. viticola were anticipated to have already formed by 48–72 hpi (Unger et al., 2007; Yin X. et al., 2017). As shown in Figure 10, at 96 hpi, 11 out of 27 PvCRN genes were expressed at relatively high levels, and these included PvCRN10, PvCRN14, PvCRN16, PvCRN17, PvCRN18, PvCRN30, PvCRN35, PvCRN2, PvCRN6, PvCRN25, and PvCRN29. To identify more PvCRN genes that were significantly transcribed during infection, RT-PCR was conducted with the mixed mRNA from inoculated grapevine leaves at 72 and 120 hpi. The mRNAs for a few PvCRN genes were detectable by RT-PCR at 72 hpi. Transcripts of PvCRN7, PvCRN9, and PvCRN21, which are PvCRNs with signal peptides, were detectable at 72 hpi rather than 96 hpi. PvCRN14, PvCRN16, PvCRN17, and PvCRN6 continued to be expressed between 72 and 96 hpi. It is intriguing that none of the 27 PvCRN genes studied showed evident accumulation of their mRNAs at 120 hpi (Figure 10).

Taken together, PvCRN14, PvCRN16, and PvCRN17 may be the strategic effectors of P. viticola, as evidenced by their high expression levels at 72–96 hpi and their suppression of Bax-induced PCD. PvCRN23 suppressed Bax-CD and promoted the susceptibility of N. benthamiana to a semi-biotrophic pathogen P. capsici, which encodes CRN proteins with low similarities to those of P. viticola, suggesting these contribute to the pathogenicity of P. viticola. PvCRN20 is another impressive effector that suppressed plant HR-like cell death triggered by INF1 or Bax and promoted P. capsici colonization of N. benthamiana. PvCRN19 increased the pathogenicity of P. capsici despite its inability to suppress the plant HR-like cell death induced by INF1 and Bax. PvCRN10 and PvCRN26 increased resistance in N. benthamiana, which was at odds with their abilities to prevent cell death associated with plant defense responses to pathogens. Therefore, the virulence of PvCRN proteins is quite complicated to determine based on the metrics above.