Dothistroma septosporum NZE10 (de Wit et al., 2012), GUA2 and COLN (Bradshaw et al., 2019), as well as D. pini CBS 116487 (BioSample ID SAMN02254964) and F. fulva 0WU (de Wit et al., 2012), were used to identify candidate effectors for characterization in this study. Escherichia coli DH5α (Taylor et al., 1993) and Agrobacterium tumefaciens GV3101 (Holsters et al., 1980) were used for gene cloning and A. tumefaciens-mediated transient transformation assays (ATTAs), respectively. N. tabacum Wisconsin 38 and N. benthamiana were used as model non-host plants for ATTAs, while N. benthamiana was used for tobacco rattle virus (TRV)-mediated gene silencing experiments. Pinus radiata clonal shoots, derived from family seed lots that are, compared to each other, relatively susceptible (S6 and S11) or tolerant (R4) to DNB, and grown from embryogenic tissue under sterile conditions on LPch agar (Hargreaves et al., 2004), were provided by Scion (New Zealand Forest Research Institute Ltd., Rotorua, New Zealand) and used in protein infiltration assays.

Members of the Ecp20 and Ecp32 protein families were identified from the predicted proteomes and/or genomes of D. septosporum NZE10 and F. fulva 0WU (de Wit et al., 2012). Chromosome locations of the FfEcp20 and FfEcp32 genes were identified using the F. fulva Race 5 genome assembly (Zaccaron et al., 2022). Gene family members were also identified from genome sequences of 18 other D. septosporum isolates (Bradshaw et al., 2019) and D. pini CBS 116487 (GenBank assembly accession GCA_002116355.1), using reciprocal BLASTp and tBLASTn searches in conjunction with an E-value cut-off of <10⁻⁵. BLASTp was also employed to determine the level of conservation for each family member across fungal species in the Joint Genome Institute (JGI) MycoCosm database, using an E-value threshold of 10⁻⁵. EffectorP v3.0 (Sperschneider and Dodds, 2021) was used to predict whether members of the Ecp20 and Ecp32 families are effectors. All D. septosporum NZE10, D. pini CBS 116487, and F. fulva 0WU nucleotide and amino acid sequences for Ecp20 and Ecp32 family members are shown in Supplementary Table S1.

Protein tertiary structure predictions were performed using AlphaFold2 (Jumper et al., 2021; Mirdita et al., 2022), and the Dali server (Holm, 2020) was used to identify proteins with structural similarity in the Research Collaboratory for Structural Bioinformatics Protein Data Bank (RCSB PDB). Here, a Dali Z-score of 2 or greater was used to infer structural similarity. Protein tertiary structures were visualized and rendered in PyMol v2.5, with alignments carried out using the CEalign tool (DeLano, 2002).

Protein sequence alignments were performed using Clustal Ω (Sievers et al., 2011) in Jalview.¹ Phylogenetic trees were constructed with the neighbor-joining method using Geneious Software v9.1.8 (Kearse et al., 2012).

Candidate effector genes of D. septosporum NZE10, GUA2, and COLN, D. pini CBS 116487 and F. fulva 0WU were cloned in the pICH86988 ATTA expression vector, which contains the nucleotide sequence encoding the N. tabacum PR1α signal peptide for secretion into the plant apoplast, followed by a 3 × FLAG for detection via Western blotting (Weber et al., 2011), according to the method described by Guo et al. (2020) in cases where the genes had no predicted introns, or synthesized directly into pICH86988 by Twist Bioscience (San Francisco, CA, United States) in cases where the genes had predicted introns. For the genes that were cloned into pICH86988, the mature coding sequence was first amplified by PCR from genomic DNA extracted from the fungus, with genomic DNA extraction performed as described by Doyle and Doyle (1987). PCRs were performed with Phusion High-Fidelity DNA polymerase (New England Biolabs, Beverly, MA, United States), and PCR products were extracted from agarose gels using an E.Z.N.A.® Gel Extraction Kit (Omega Bio-tek, Norcross, GA, United States). PCR primers were designed with Primer3 (Untergasser et al., 2012) in Geneious v9.1.8 (Kearse et al., 2012). These primers, which were synthesized by Integrated DNA Technologies (IDT; Supplementary Table S2), had 5′ extensions containing a BsaI recognition site and four-base overhang specific for Golden Gate modular cloning (Engler et al., 2009) in between the CaMV 35S promoter and octopine synthase terminator of pICH86988. Versions of the candidate effector genes were also ligated into pICH86988 without the nucleotide sequence encoding the PR1α signal peptide to prevent secretion of the proteins they encode into the apoplast. Constructs were then transformed into E. coli, plasmids extracted using an E.Z.N.A.® Plasmid DNA Mini Kit I (Omega Bio-tek), and correct assemblies confirmed by sequencing. ATTA expression vectors were transformed into A. tumefaciens by electroporation as described previously (Guo et al., 2020).

To heterologously express candidate effector genes in N. benthamiana and N. tabacum using ATTAs, single colonies of A. tumefaciens carrying pICH86988 constructs of interest were first inoculated into selective lysogeny broth (LB) containing 50 μg/ml kanamycin, 10 μg/ml rifampicin, and 30 μg/ml gentamycin, then incubated overnight at 28°C. Cells were subsequently collected by centrifugation at 2,500 g for 5 min and resuspended in 1 ml of infiltration buffer [10 mM MgCl₂:6H₂O, 10 mM MES-KOH (Sigma-Aldrich, St. Louis, MO, United States), 100 μM acetosyringone (Sigma-Aldrich)] to an OD₆₀₀ of 0.5. Following resuspension, the abaxial surface of N. benthamiana and N. tabacum leaves was infiltrated with the appropriate A. tumefaciens suspension using 1 ml needleless syringes. Here, the extracellular elicitin INF1 from Phytophthora infestans, which is recognized by the LRK1 immune receptor in Nicotiana species (Kamoun et al., 1997; Kanzaki et al., 2008), was used as a positive cell death control, while the empty pICH86988 vector was used as a negative no cell death control. At least 12–24 infiltration zones were tested for each candidate effector gene or control (consisting of two leaves from each of two plants × 3–6 repeat experiments). Plants were monitored for up to 7 days, and photographs taken using a Nikon D7000 camera. Infiltration zones were assessed on whether the protein tested elicited a strong or weak cell death response, or no cell death. A strong cell death response was scored when it was indistinguishable from the response elicited by INF1 and around the entire infiltration zone, while a weak response was considered when the protein elicited a weaker response across approximately 50% of the infiltration zone when compared with INF1. A no-cell death response was indicated when the infiltration zone had the same response as the negative empty pICH86988 vector control.

To confirm that candidate effector proteins had been produced in N. benthamiana and N. tabacum leaves using ATTAs, and to determine whether these proteins were of the predicted size, Western blotting was conducted with total protein as described previously by Guo et al. (2020). For protein separation by SDS-PAGE, 12% separating and 5% stacking gels were used, with proteins transferred to PVDF membranes. Anti-FLAG® M2 primary antibody produced in mouse (Sigma-Aldrich), together with anti-mouse secondary antibody produced in chicken (Santa Cruz Biotechnology, Dallas, TX, United States) and SuperSignal® West Dura Extended Duration substrate (Thermo Fisher Scientific, Waltham, MA, United States), were used for protein detection. Proteins were visualized using an Azure Biosystems c600 Bioanalytical Imaging system (Azure Biosystems, Dublin, CA, United States).

Genes encoding candidate effector proteins were amplified by PCR as above from the previously generated ATTA expression vectors, without their native signal peptide sequences. Here, the 5′ PCR primers incorporated a FLAG-tag for detection by Western blotting. PCR primers are shown in Supplementary Table S2. PCR amplicons were gel-extracted as above and ligated, using DNA ligase (New England Biolabs), into the SmaI/EcoRI restriction sites of the expression vector pPic9-His₆ (Invitrogen, Carlsberg, CA, United States), which encodes an α-factor signal peptide for protein secretion and a hexa-histidine (6 × His) tag for protein purification. The ligated products were then transformed into E. coli. The expression constructs were sequenced and contained the predicted mature sequence of the candidate effectors fused to a 6 × His tag at their N-terminus. The expression cassette was linearized with SacI or SalI (New England Biolabs) restriction enzymes and transformed into Pichia pastoris GS115, according to Kombrink (2012).

Protein expression in P. pastoris was performed according to Weidner et al. (2010). Briefly, protein expression was induced in 200 ml of BMMY (Buffered Methanol-complex Medium), through the addition of methanol to a concentration of 0.5% (v/v) every 24 h, for 72 h. The cells were then removed by centrifugation at 4,500 g for 30 min and the supernatant filter-sterilized by passage through a 0.22 μm membrane (ReliaPrep, Ahlstrom, Helsinki, Finland). Finally, the pH of the sterilized culture filtrate was adjusted to 8 with NaOH.

The secreted candidate effector protein (fused with an N-terminal 6 × His tag) was purified from culture filtrate using immobilized metal ion affinity chromatography (IMAC), in conjunction with a Ni Sepharose™ 6 Fast Flow (GE Healthcare, Chicago, IL, United States) column, according to the manufacturer’s protocol. Before loading, the column (Glass Econo-Column®—BioRad, Hercules, CA, United States) was packed with 5 ml of resin and equilibrated by washing with binding buffer (20 mM sodium phosphate, 0.5 M NaCl, pH 7.4). The culture filtrate was added to the column at a flow rate of 1 ml/min, and protein eluted with an elution buffer (20 mM sodium phosphate, 0.5 M NaCl and 500 mM imidazole, and pH 7.4). The elution fractions were mixed, and elution buffer was added to a final volume of 50 ml to obtain sufficient volume for vacuum-infiltration of pine shoots. Western blotting was performed as above to determine the presence of each candidate effector protein in P. pastoris culture filtrate and after purification. Protein concentrations in culture filtrates and purified solutions were determined by using a Pierce™ Coomassie (Bradford) Protein Assay Kit (Thermo Fisher Scientific), as per the manufacturer’s protocol.

Proteins were infiltrated into tissue-cultured pine shoots at a concentration of approx. About, 20 μg/ml using the method developed by Hunziker et al. (2021). Briefly, clonal rootless pine shoots were maintained in LPch agar (Hargreaves et al., 2004) in glass jars, with each jar containing 6–8 shoots. For this experiment, clonal shoots originating from two relatively Dothistroma-susceptible families (S6 and S11) and one relatively Dothistroma-tolerant family (R4; based on comparative field data) were used. The shoots were completely submerged in approx. 50 ml of each purified candidate protein or control solution (positive control: DsEcp32-3; negative control: elution buffer). Samples were exposed to vacuum in a glass chamber for 5 min to facilitate protein uptake. Shoots were then briefly rinsed in sterile MilliQ (MQ) water and subsequently placed back into the LPch agar in the glass jar. The experiment was conducted at 22°C in a room with natural light. Photos were taken 7 days after infiltration (dai) using a Nikon D7000 camera.

Fragments used to generate virus-induced gene silencing (VIGS) constructs were amplified by PCR from cDNA or genomic DNA of N. benthamiana using PrimeSTAR GXL DNA Polymerase (Takara Bio Inc.) with the primers listed in Supplementary Table S2. The TRV VIGS system is composed of pTRV-RNA1, which encodes replication related proteins, and pTRV-RNA2, which harbors the viral coat protein, and the candidate fragment sequences that target silencing to the corresponding genes (MacFarlane, 1999; Liu et al., 2002). The purified fragments were cloned into the pTRV-RNA2 vector (Dong et al., 2007) using the ClonExpress II One Step Cloning Kit (Vazyme Biotech Co., Ltd., Nanjing, China). The resulting vectors were verified by sequencing and individually transformed into A. tumefaciens strain. DsEcp20-3 was screened against around 100 TRV VIGS constructs which targeted different N. benthamiana LRR-RLP and LRR-RLK-encoding genes. Information on this library of silencing constructs can be found in Supplementary Data 1 from Wang et al. (2018). Primers for T26, T26-2, TRV:BAK1 (Heese et al., 2007), and TRV:SOBIR1 (Liebrand et al., 2013) are shown in Supplementary Table S2.

Agrobacterium tumefaciens strains carrying N. benthamiana gene silencing vectors (Wang et al., 2018) were grown overnight in LB medium with the appropriate antibiotics. A. tumefaciens cells were pelleted as mentioned above, resuspended, and incubated in infiltration medium (10 mM 2-[N-morpholino] ethanesulfonic acid pH 5.6, 10 mM MgCl₂, and 200 μM acetosyringone) for 3–4 h before agroinfiltration. A. tumefaciens cultures expressing pTRV-RNA2 constructs were mixed in a 1:1 ratio with A. tumefaciens culture expressing pTRV1 to a final OD₆₀₀ of 1.0 before infiltration into cotyledons of four-leaf-stage N. benthamiana seedlings. Phytophthora sojae NPP1 and XEG1 (Fellbrich et al., 2002; Ma et al., 2015), and P. infestans INF1 were used as cell death-eliciting controls for VIGS. XEG1-and INF1-triggered cell death was known to be compromised in N. benthamiana leaves treated with TRV:BAK1 or TRV:SOBIR1, but not with TRV:GFP, which was used as a negative control (Wang et al., 2018).

To determine the expression levels of BAK1, SOBIR1, and T26 in the TRV-mediated VIGS experiment, N. benthamiana leaves of similar size were selected and ground to a powder in liquid nitrogen. Total RNA was isolated from 100 mg of ground material using the E.Z.N.A.® Total RNA Kit I (Omega Bio-tek) and used as a template for first strand cDNA synthesis with PrimeScript Reverse Transcriptase (Takara Bio Inc., Kusatsu, Shiga, Japan). Quantitative reverse transcription PCR (qRT-PCR) was performed on an ABI 7500 Fast Real-Time PCR system (Applied Biosystems Inc., Foster City, CA, United States) using PrimeScript RT Master Mix (Takara Bio Inc.) with translation elongation factor 1 alpha (EF-1α) as an endogenous control and the primers listed in Supplementary Table S2. Data were analyzed using the 2^−ΔΔCT method (Livak and Schmittgen, 2001).

In our previous work, two small, secreted candidate effector proteins from D. septosporum NZE10, Ds70694 and Ds70057, triggered cell death in the non-host species N. tabacum and N. benthamiana (Hunziker et al., 2021). Both proteins are homologs of Ecps from F. fulva and are part of two different protein families, Ecp20 and Ecp32, respectively, that were shown to be located in the apoplast of tomato leaves infected with F. fulva (Mesarich et al., 2018). More specifically, Ds70694 is an ortholog of FfEcp20-3 from F. fulva and was therefore named DsEcp20-3, while Ds70057 is an ortholog of FfEcp32-3 and was therefore named DsEcp32-3.

The Ecp20 family has four paralogues in both species, all with signal peptides (Supplementary Figures S1, S2) and no known functional domains. Based on sequence similarities between them, DsEcp20-3 and FfEcp20-3, together with the other orthologous pairs Ds/FfEcp20-1, Ds/FfEcp20-2, and Ds/FfEcp20-4, were grouped together (Figure 1A, Supplementary Figure S1 A). Interspecies pairwise amino acid identities between all family members from D. septosporum and F. fulva ranged from 10.6 to 85.5%, while intraspecies pairwise identities between all family members ranged from 14.2 to 40.9% in D. septosporum and 11.8 to 44.2% in F. fulva (Supplementary Table S3). The Ds/FfEcp20-1 genes contain one exon, with the intron located at the same position, while genes Ds/FfEcp20-2, -3, and -4 do not have any introns (Supplementary Figure 2 A). All genes encode a protein with four cysteine residues, although the location of these varies slightly from protein pair to protein pair (Supplementary Figures S1 A, S2A).

Based on data from a previously published transcriptome study (Bradshaw et al., 2016), all genes of the D. septosporum Ecp20 family are expressed during pine infection, with DsEcp20-3 and DsEcp20-4 being the two most highly expressed (Supplementary Table S4). DsEcp20-3 showed strong up-regulation in the Mid and Late in planta stages that mark the start and end of necrotrophic infection, when compared with the Early (biotrophic) stage and in vitro, while DsEcp20-4 is strongly upregulated in the Late in planta stage. For the corresponding genes in F. fulva during tomato infection, as based on data from a previously published transcriptome study (Mesarich et al., 2018), FfEcp20-2 and FfEcp20-3 were highly expressed throughout the timepoints analyzed, while FfEcp20-4 was highly expressed at the beginning of infection and downregulated at later time points, when compared with in vitro (Supplementary Table S4). It should be noted that FfEcp20-1 had no gene model and therefore had no FPKM value assigned to it in the previous study (Mesarich et al., 2018). Thus, no expression information was available for this gene.

The Ds/FfEcp32 family was analyzed in the same way as the Ds/FfEcp20 family above. The Ecp32 family has five paralogues in F. fulva, named FfEcp32-1 to-5, and four in D. septosporum, named DsEcp32-1 and DsEcp32-3 to-5 (there is no ortholog of FfEcp32-2 in D. septosporum; Figure 1B; Supplementary Figure S1 B). All these family members have signal peptides (Supplementary Figures S1, S2) and no known functional domains. Interspecies pairwise amino acid identity between all family members from D. septosporum and F. fulva ranged from 10.6 to 82%, while intraspecies pairwise identity between members ranged from 22.1 to 42.1% in D. septosporum and 21.6 to 49% in C. fulvum (Supplementary Table S3). Regarding the exon-intron structure, Ds/FfEcp32-1, FfEcp32-2, and Ds/FfEcp32-3 do not have any introns. In contrast, the Ds/FfEcp32-4 gene pair has one intron at the same position, while the Ds/FfEcp32-5 gene pair has two introns at the same positions (Supplementary Figure S2 B). In terms of cysteine residues, the Ds/FfEcp32 proteins have anywhere from none (Ds/FfEcp32-5) to five (Ds/FfEcp32-4), although in the case of DsEcp32-3, an additional cysteine is present compared to the Ff ortholog (Supplementary Figures S1 B, S2B).

All DsEcp32 genes are also expressed during pine infection. However, only DsEcp32-3 and DsEcp32-5 are highly expressed in planta, especially at the Mid and Late necrotrophic stages when compared with in vitro. DsEcp32-1 and DsEcp32-4 are only expressed at a low level in planta. During F. fulva infection of tomato, FfEcp32-1, FfEcp32-2, and FfEcp32-3 are highly expressed at all timepoints. FfEcp32-4 was not expressed under any condition, while FfEcp32-5 was only expressed at 12 dpi, and was downregulated compared with in vitro (Supplementary Table S4).

Genomes from the two species are mesosyntenic (de Wit et al., 2012; Zaccaron et al., 2022) and, in keeping with this, we found conservation of chromosome location between the two species for each of the Ds/FfEcp20 and Ds/FfEcp32 gene pairs (Supplementary Figure S3). Taken together, sequence similarity, intron position and chromosome localization suggest a common evolutionary origin for the Ecp20 and Ecp32 genes from both species.

We predicted the tertiary structures of all Ecp20 family members from D. septosporum NZE10 and F. fulva 0WU using AlphaFold2 (Figure 2; Supplementary Figure S4). All proteins from the Ecp20 family are predicted to be structurally similar to the characterized β-barrel fold of Alt a 1 from Alternaria alternata (RCSB protein data bank [PBD] ID: 3V0R; Chruszcz et al., 2012; Supplementary Tables S4, S5). Figure 2 shows the predicted structures for DsEcp20-3 and DsEcp20-4, as well as the alignments between DsEcp20-3 and the characterized structure of Alt a 1 from A. alternata and between the predicted structures of DsEcp20-3 and DsEcp20-4. The predicted structures for all the DsEcp20 and FfEcp20 proteins are shown in Supplementary Figure S4 along with characterized crystal structures of similar proteins from other fungal species.

The tertiary structure of Ecp32 proteins was also predicted using AlphaFold2 (Figure 3, Supplementary Figure S5). All Ecp32 family members had significant hits with trypsin inhibitors and lectins; these are proteins that share a β-trefoil fold and are known virulence factors in plant-pathogenic fungi (Renko et al., 2012; Varrot et al., 2013; Sabotič et al., 2019; Supplementary Table S5). Figure 3 shows the predicted structures for DsEcp32-3 and DsEcp32-4, as well as the alignments between DsEcp32-3 and the characterized structure of a trypsin inhibitor from the plant species Enterolobium contortisiliquum (RCSB PBD ID: 4J2K; Zhou et al., 2013), which is the top hit in the PDB for most Ds/FfEcp32 proteins. The alignment between the predicted structures of DsEcp32-3 and DsEcp32-4 is also shown in Figure 3, while all the predicted Ds/FfEcp32 structures can be found in Supplementary Figure S5 along with characterized structures of similar proteins.

The Ecp20 family is present in fungal species from other classes. DsEcp20-1 had 27 significant BLASTp hits in the JGI MycoCosm protein database. Two of these were from other Dothideomycete species (including the ortholog in F. fulva), while the rest were with from species in the Eurotiomycetes class (Supplementary Table S6). DsEcp20-2 and DsEcp20-3, the most closely related to each other among the DsEcp20 family members (Figure 1A), both had the same 28 hits in JGI. All of these matches were with other Dothideomycete species and, of these, 24 were hits to proteins from other plant pathogens (Supplementary Table S6). DsEcp20-4 had 1,006 hits in the JGI MycoCosm protein database, of which 241 were proteins from plant pathogens (Supplementary Table S6).

In general, the Ecp32 family is more widely conserved among other fungal species compared to the Ecp20 family. Individual searches in the JGI MycoCosm protein database for each of the DsEcp32 family members yielded approximately 1,000 hits for each family member (Supplementary Table S6). Those hits included proteins from fungal species with different lifestyles, ranging from saprophytes, to biotrophs, hemibiotrophs, and necrotrophs. For example, DsEcp32-3 had 158 hits to proteins from fungal plant pathogens in JGI. Of these, 42 were with other members from the Mycosphaerellaceae family. These included Cercospora zeae-maydis with four paralogs, Pseudocercospora (Mycosphaerella) eumusae with six, Pseudocercospora fijiensis with five, and Zymoseptoria tritici with three paralogs (Supplementary Table S6).

Dothistroma pini is also a causal agent of DNB (Barnes et al., 2004). Thus, we searched for orthologs of DsEcp20/32 encoded in the D. pini genome. All proteins from the D. septosporum Ecp20 and Ecp32 families had orthologs in the genome of D. pini, except Ecp32-5, for which the gene encoding this protein appears to be a pseudogene. The protein sequences were very similar between family members from the two Dothistroma species, with full-length pairwise amino acid identity varying from 81.1 to 93.8% (Supplementary Tables S1, S3).

We next addressed the question of whether the D. septosporum Ecp20 and Ecp32 family genes are conserved in other isolates of this species and whether they are identical in sequence to NZE10. Genome sequences were available from 18 D. septosporum isolates collected from different geographic locations (Bradshaw et al., 2019). All members of the D. septosporum Ecp20 and Ecp32 families were present in the 18 other genomes. In each of these genomes, there was at least one single nucleotide polymorphism (SNP) leading to an amino acid difference in at least one family member for both Ecp20 and Ecp32 (Supplementary Table S7).

A total of 59 different SNPs were identified among the Ecp20 family members across the 18 D. septosporum isolates, compared to NZE10 (Figure 4). DsEcp20-1, DsEcp20-2, and DsEcp20-4 had approximately the same number of SNPs: 11, 13, and 15, respectively, while DsEcp20-3 had a total of 20. For DsEcp20-1, DsEcp20-3, and DsEcp20-4, however, most SNPs (61, 60, and 60%, respectively) were synonymous. In the case of DsEcp20-2, however, only 30% were synonymous and a higher number of SNPs leading to an amino acid difference (nine out of 13; 70%) were observed (Figure 4A). The Ecp32 gene family had a higher number of DNA modifications across the 18 D. septosporum genomes analyzed, with a total of 117 SNPs and three insertions. The two most polymorphic genes were DsEcp32-3 and DsEcp32-5. Interestingly, while DsEcp32-3 had 31 sequence modifications, only five of these led to an amino acid difference (16%). All other family members had nine or 10 SNPs leading to an amino acid difference (Figure 4B).

The Guatemalan (GUA1 and GUA2) and Colombian (COLN and COLS) isolates showed the greatest amino acid sequence diversity, when compared to strain NZE10, for both the Ecp20 and Ecp32 family members. For DsEcp20-1, the most diverse isolates were COLN and COLS, with identical sequences, while COLN had the most sequence variation in DsEcp32-1. For DsEcp20-2 and-4, and Ecp32-3, -4, and-5, both Guatemalan isolates were the most diverse and, for DsEcp20-3, GUA2 had greater amino acid diversity (Supplementary Table S7).

In a previous study, DsEcp20-3 and DsEcp32-3 were shown to elicit cell death in the non-host plants N. benthamiana and N. tabacum (Hunziker et al., 2021); these two model plant species have been used in other studies of plant-pathogen interactions and their responses to the same protein sometimes vary (Guo et al., 2020; Hunziker et al., 2021). Thus, we set out to determine whether other family members of both D. septosporum and F. fulva could elicit the same response in these hosts. To achieve this, all Ecp20 and Ecp32 family members were transiently expressed in N. benthamiana and N. tabacum using ATTAs, and the percentages of plant responses in three different categories recorded: strong cell death, weak cell death and no cell death (Figure 5; Supplementary Figure S6). Here, orthologous pairs from both pathogens were tested on the same leaf, so that differences between them could be better assessed.

As expected, the positive control INF1 triggered strong cell death responses and the negative control empty vector did not elicit a response. Among Ecp20 family members, FfEcp20-2 triggered cell death responses in N. benthamiana in 91.7% of infiltration zones, while its ortholog DsEcp20-2 did not elicit any cell death responses. Both FfEcp20-3 and DsEcp20-3 caused consistent plant cell death in N. benthamiana, with DsEcp20-3 having 58% of infiltration zones showing strong cell death across the entire infiltration zone (Figures 5A,B). In N. tabacum, DsEcp20-3 was the only family member that triggered a cell death response (Supplementary Figure S6). Interestingly, DsEcp20-4, one of the most highly expressed secreted proteins during infection of pine by D. septosporum (Supplementary Table S4; Bradshaw et al. (2016)), and its ortholog from F. fulva, FfEcp20-4, did not induce cell death responses in either N. benthamiana or N. tabacum (Figure 5; Supplementary Figure S6).

The ability of Ecp32 family members to trigger cell death was also assessed in both Nicotiana species. In this case, all proteins from the Ecp32 family, apart from the Ds/FfEcp32-4 protein pair, induced some degree of cell death in both plant species. Interestingly, DsEcp32-1 consistently triggered cell death in N. benthamiana, while its ortholog from F. fulva, FfEcp32-1, only caused a strong cell death response in 3.5% of infiltration zones across all replicates (Figures 5C,D). In contrast, in N. benthamiana, FfEcp32-3 was capable of triggering consistent strong cell death responses, while DsEcp32-3 only trigged a strong response in approximately 20% of infiltration zones. The Ds/FfEcp32-5 pair triggered similar degrees of cell death responses in N. benthamiana (Figure 5D).

To determine whether apoplastic localization is required for members of the Ecp20 and Ecp32 families to trigger cell death, proteins that previously elicited a plant response, but lacked the PR1α signal peptide for secretion to the apoplast, were transiently expressed in N. benthamiana. Without the signal peptide, none of the proteins tested were able to cause cell death (Supplementary Figures S7, S8).

In cases where a cell death response was not observed at all, or where only weak cell death was observed, a Western blot analysis was performed to confirm the presence of the proteins in the tissue of the infiltrated plants (Supplementary Figure S8). All proteins that did not trigger cell death or only triggered a weak cell death response were detected via Western blot, indicating that the lack of cell death response was not due to absence of those proteins.

Taken together, these results indicate that Ecp20 and Ecp32 family members from two closely related fungal pathogens, D. septosporum and F. fulva, differ in their ability to induce a cell death response in non-host species. Furthermore, the finding that proteins without a signal peptide failed to trigger a plant cell death response, suggests that secretion into the apoplastic environment of N. benthamiana is essential for their cell death-eliciting activity.

Alleles of some Ecp20 and Ecp32 family genes identified from D. pini, and from isolates of D. septosporum other than NZE10, were tested for the ability of their encoded proteins to trigger cell death in N. benthamiana. For the proteins from D. septosporum NZE10 that consistently triggered the highest percentages of cell death (DsEcp20-3, DsEcp32-1, and DsEcp32-3), we tested orthologs of other D. septosporum alleles and found they triggered the same responses as those of D. septosporum NZE10 (Supplementary Figure S9). The same was true for the respective orthologs of DpEcp20-3, DpEcp32-1, and DpEcp32-3 from D. pini, which also elicited cell death responses in N. benthamiana. These results suggest conservation of cell death-eliciting activity between isolates of D. septosporum and between the two Dothistroma species for members of the Ecp20 and Ecp32 families.

The pine infiltration method described in Hunziker et al. (2021) proved to be a suitable approach for testing the cell death-eliciting activity of candidate apoplastic effector proteins from pine pathogens in P. radiata. We previously showed that DsEcp32-3 triggered cell death in three P. radiata genotypes (S6, S11, and R4). Using the same method, we tested two additional proteins from the DsEcp20 family, DsEcp20-3 and DsEcp20-4, and repeated the infiltration of DsEcp32-3 as a positive cell death control (Figure 6; Supplementary Figure S10; Hunziker et al., 2021).

The DsEcp32-3 positive control triggered cell death in all three pine genotypes, as expected (Figure 6). Infiltration of elution buffer (negative control) did not cause any damage or visible stress responses. Interestingly, both tested proteins gave the same response as they had shown in the non-host Nicotiana plants: DsEcp20-3 consistently elicited cell death in the same three pine genotypes used in the previous study, while DsEcp20-4 failed to elicit any response in the pine shoots.

To determine how cell death elicitors from the Ds/FfEcp20 and 32 families were perceived by plants, we silenced genes encoding the extracellular receptor-like kinase co-receptor genes BAK1 and SOBIR1, as well as leucine-rich repeat (LRR) receptor-like genes, in N. benthamiana (Figure 7), in conjunction with agro-infiltration, as previously described by Wang et al. (2018). As expected, agro-infiltration of positive controls NPP1, INF1, and XEG triggered consistent strong cell death responses in N. benthamiana leaves treated with TRV:GFP, while INF1-and XEG1-triggered cell death was compromised in leaves treated with TRV:BAK1 or TRV:SOBIR1. We also consistently found that DsEcp20-3-triggered cell death was compromised in N. benthamiana leaves in which the receptor-like kinase genes BAK1 or SOBIR1 were silenced (Figure 7B). In addition, we found that DsEcp20-3-triggered cell death was compromised in N. benthamiana leaves treated with TRV VIGS constructs TRV:T26 and TRV:T26-2 (Wang et al., 2018; Figure 7D). TRV:T26 and TRV:T26-2 both target the same two N. benthamiana genes but in different regions of those genes. One of these target genes encodes an LRR-type extracellular receptor-like protein (Sol Genomics Network accession number—Niben101Scf03138g01049.1) and one an extracellular LRR (Niben101Scf01834g02009.1). We further screened other members of the Ecp20 and Ecp32 families (FfEcp20-3, DsEcp32-3, and FfEcp32-3), and found that cell death triggered by FfEcp20-3 and FfEcp32-3 was also significantly compromised in N. benthamiana leaves treated with TRV:BAK1, TRV:SOBIR1 or either of the T26 constructs (Figures 7B,D). For DsEcp32-3-triggered cell death, we did not detect any reduction on TRV:BAK1- or TRV:SOBIR1-silenced plants (data not shown). Taken together, these results suggest that the cell death responses triggered by members of the Ecp20 and Ecp32 families from D. septosporum and F. fulva in N. benthamiana are the result of recognition by extracellular immune receptors, and that the transduction of defense response signals following this recognition requires the extracellular co-receptors BAK1 and SOBIR1.