Introduction

Pests and pathogens are a persistent threat to plant health1, and this is likely to become worse with warming climate2. Forest trees, which provide important ecosystem functions and renewable resources, and have a role in mitigating global climate change3, are not exempt from this threat. Nonetheless, these functions are often undervalued4, and gymnosperm pathology research, particularly at the molecular level, lags behind that of short-lived angiosperm crop plants5.

Dothistroma needle blight (DNB) is one of the most destructive foliar diseases of pine trees6. It has been reported in 76 countries, affecting 109 different host species6. DNB is caused by the Dothideomycete fungi Dothistroma septosporum (Dorogin) M. Morelet and D. pini Hulbary6. These fungi colonise and cause lesions on the needles, triggering premature defoliation which can result in reduced growth and sometimes death of trees6,7. D. septosporum is responsible for most occurrences of DNB, particularly in North America, Europe and across the Southern Hemisphere, where it has devastated commercial plantations of Pinus radiata8. The increased incidence and severity of DNB seen over the last two decades has been attributed to both natural and anthropogenic causes, including changes in climate9,10. Existing DNB management strategies can counteract the disease in some cases, but huge losses are still seen in heavily affected regions, including some with native pine forests6,8.

To counteract any pathogen, it is important to understand how the pathogen achieves compatible interactions (infections) with its host plant(s). Of central importance to this compatibility is the host apoplast, where early contact between plant and pathogen cells is made11. For invading pathogens, the apoplast is a hostile environment, with constitutively produced plant molecules, such as secondary metabolites and hydrolytic enzymes, that impede growth12. In addition to these defences, the apoplast is monitored by cell surface-localised immune receptors, termed pattern-recognition receptors (PRRs). PRRs recognise foreign or ‘damaged self’ molecules, collectively called invasion patterns (IPs), to activate the plant's innate immune system13. Following recognition by these PRRs, a series of immune responses of varying intensity are triggered, the strongest of which is the hypersensitive response (HR)14, which involves localised cell death and a burst of reactive oxygen species (ROS) that quickly halt the invading pathogen’s growth. Given this hostility, successful pathogens must neutralise the apoplastic environment to colonise their hosts and cause disease.

The ability to manipulate plant defences in the apoplast using secreted molecules is widely shared among microbes, including fungi15. These molecules, usually small proteins, are called effectors (virulence factors) and can evolve rapidly. However, following adaptation of the host, effectors that serve a virulence function for the pathogen may be recognised as IPs by host immune receptors and thus elicit resistance responses11. Immune receptors can also be located inside plant cells, where some effectors are delivered to perform their virulence functions. Intriguingly, some pathogens with necrotrophic stages in their lifecycles exploit plant cell death associated with IP recognition16,17. In these cases, the hosts’ attempts to prevent invasion instead favour pathogen growth by initiating cell necrosis, which provides nutrition for the pathogen.

Plant immune receptors that provide resistance against pathogens are encoded by resistance genes, and immune receptors that are hijacked by pathogens to provide susceptibility are encoded by susceptibility genes. The discovery of resistance and susceptibility genes in plants has averted yield losses for several crop species by enabling resistant cultivars to be selected17. Fuelled by ever-growing genome, transcriptome and proteome resources, the field of ‘effectoromics’, in which pathogen effectors are screened for those that elicit or suppress plant defence, has enabled the identification of hosts with resistance and susceptibility genes in a growing number of plant species16,17,18,19. Furthermore, these studies improve our understanding of host–pathogen interactions that will ultimately lead to new, more targeted approaches to disease control.

Published research focussing on foliar pine pathogen effectors is scarce, with no efficient screening methods established involving the host species. In the case of D. septosporum, inroads have been made based on the identification of effector homologues from the well-studied tomato leaf mould pathogen, Cladosporium fulvum which, like D. septosporum, colonises the plant apoplast and does not produce specialised infection structures such as haustoria. Indeed, research has shown that D. septosporum and C. fulvum share several functional apoplastic effectors, with one such example being the orthologous effector pair DsEcp2-1 (D. septosporum) and Ecp2-1 (C. fulvum)20,21,22. With regards to C. fulvum, Ecp2-1 has been shown to trigger HR in the presence of the corresponding Cf-Ecp2 immune receptor in tomato (identity not yet known), suggesting that it is recognised as an avirulence determinant23,24. The same effector has also been found to elicit cell death in Nicotiana spp., including N. tabacum, following its recognition by an immune receptor in these non-hosts (identity also not yet known)5,24. In D. septosporum, a similar story appears to be emerging, with DsEcp2-1 shown to trigger cell death in Nicotiana tabacum25. This effector has also recently been implicated as a possible avirulence determinant, with mutants of D. septosporum deleted for the DsEcp2-1 gene found to cause larger lesions than wild-type in pine25. Notably, small secreted proteins from the nectotrophic forest pathogen, Heterobasidion annosum, have also been shown to elicit cell death in N. benthamiana26. Thus, non-host angiosperm plants appear to recognise effector proteins from gymnosperm pathogens in a highly conserved fashion.

Here, we focused on candidate apoplastic effectors of D. septosporum that might be involved in a successful infection and/or triggering host defence. The specific aims of our study were to (a) functionally characterise D. septosporum candidate effectors (DsCEs) based on recognition by conserved PRRs in non-host model plants, and (b) develop and provide proof of concept for a new effector protein screening method in pines, employing vacuum infiltration-mediated delivery into small tissue-culture pine shoots.

Results

Dothistroma septosporum candidate effectors have sequence and structural similarity to fungal virulence factors

The D. septosporum genome has approximately 12,580 predicted genes, 397 of which encode putatively secreted proteins that are expressed during infection of P. radiata seedlings20,27,38. From this resource, a short list of 30 predicted apoplastic D. septosporum candidate effector proteins (DsCEs) was identified for further analysis (Fig. 1, Supplementary Table S1).

Figure 1
figure 1

Cloned Dothistroma septosporum candidate effectors. Description and expression of the 30 shortlisted D. septosporum effector candidates used in this study. aProtein ID numbers refer to those at JGI (https://mycocosm.jgi.doe.gov/Dotse1/Dotse1.home.html). An asterisk indicates a non-apoplastic localisation prediction according to EffectorP3.0. bFunction predictions were based on BLASTp (E < 1e−05) and HHpred. A dagger indicates that no sequence homologues were found in other foliar pine pathogens for which predicted proteomes were available. cMature protein length (predicted N-terminal signal peptide removed) in amino acids; number of cysteine residues shown in brackets. dD. septosporum gene expression in culture (FM; fungal mycelium) and in planta (Pinus radiata) at Early, Mid, Late infection stages27. Left, Reads Per Million per Kilobase (RPMK); right, heatmap representing fold (log2) changes relative to FM expression.

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We first determined if the DsCE sequences were conserved in other fungi with available genomes. All but five of the shortlisted DsCEs (i.e. Ds19040, Ds24625, Ds72737, Ds73520 and Ds74875) had homologues in at least 10 different fungal species, while nine were predicted to have conserved functional domains (Supplementary Table S2). Furthermore, almost all of the DsCEs had a homologue in at least one of the nine pine needle-associated fungi investigated (Supplementary Table S3), with more than half of these possessing a homologue in both a pathogenic species and the pine endophyte species Lophodermium nitens. Only three DsCEs (Ds19040, Ds72737 and Ds73520) appeared to be exclusive to D. septosporum, with no apparent homologues present even in the queried pine needle-associated fungi. Ds52422 was unique in having no homologues in the pathogenic species, but one homologue in L. nitens (Supplementary Table S3).

Structural analysis was carried out because some effector protein families share structural similarity, rather than sequence similarity, with effectors from different fungi28,29. HHpred analysis inferred structural relationships for five of the DsCEs that had no sequence-based functional annotations. Of these five, Ds43416 and Ds70694 showed structural similarity to Alt a 1 allergen/PevD1-like proteins, Ds52422 to endoglucanase proteins, and both Ds68958 and Ds72870 to polyketide cyclase proteins (Supplementary Table S1, Fig. 1). Notably, the gene encoding Ds43416 (Alt a 1/PevD1-like) was one of the most highly expressed and up-regulated of the DsCEs, with a remarkably high level of expression at the late infection stage (Fig. 1, Supplementary Table S1). Among the other DsCEs assessed using HHpred, possible structural relationships were also identified with proteins that carry functional domains associated with fungal virulence, namely chitin binding (lectin), cerato-platanin (sub-group of endoglucanase-like proteins) and CAP family domains (Supplementary Table S1)30,31,32. In several cases, structural relationships inferred from HHpred corroborated functional annotations that were previously predicted for several DsCEs based on sequence similarity (Supplementary Table S1).

Taken together, the short list of 30 DsCE proteins (Fig. 1) included 13 with sequence and/or predicted structural similarity to fungal proteins with described functions, and 17 with sequence similarity to yet undescribed proteins encoded in other fungal genomes (Fig. 1, Supplementary Tables S1 and S2). Twenty-five of the 30 DsCEs had at least four cysteine residues (Fig. 1), consistent with the need for disulphide bonds for stability in the hostile apoplast22. Further, an overview of the D. septosporum reference genome, which is assembled to chromosome level20, showed no particular clustering of the 30 DsCE genes on small chromosomes or near repetitive elements (Supplementary Fig. S1, Supplementary Table S1).

Six candidate effectors of D. septosporum induced cell death in non-host plants

In order to identify DsCEs that elicit cell death and are potentially recognised by non-host plant immune receptors, the 30 DsCE genes listed in Fig. 1 were transiently expressed in N. tabacum and N. benthamiana. In addition to DsEcp2-1, five other DsCEs induced cell death, indicative of a hypersensitive defence response (HR) (Fig. 2).

Figure 2
figure 2

Dothistroma septosporum NZE10 candidate effectors (DsCEs) induce cell death responses in non-host plants. Genes encoding DsCEs, with a PR1α signal peptide for secretion to the plant apoplast, were introduced into Nicotiana tabacum (Nt; top row of images) and N. benthamiana (Nb; middle row of images) by agro-infiltration. Cell death responses were observed at 6 days post-infiltration. Representative images are shown, along with the percentages of all infiltrations where cell death occurred (n = 12–24 infiltration zones, from at least three independent experiments). Ds43416 is included as a representative of DsCEs that did not elicit a cell death response in either of the plant species tested. The bottom row of images shows the results of agro-infiltration of DsCE proteins lacking a PR1α signal peptide (ΔSP; no secretion into the apoplast) in N. tabacum (DsEcp2-1, Ds70694, Ds70057, Ds71487 and Ds131885) or N. benthamiana (Ds74283). INF1, Phytophthora infestans elicitin positive control; EV, empty vector negative control.

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The DsCEs differed in their ability to consistently elicit a response in N. tabacum. Three (DsEcp2-1, Ds70057 and Ds131885) caused cell death in at least 90% of the infiltration spots within 6 days post-infiltration (dpi), while Ds70694 (66%) and Ds71487 (58%) were less consistent (Fig. 2). To gauge whether the culture density of infiltrated A. tumefaciens played a role in plant responses to the DsCEs, we trialled Agrobacterium tumefaciens-mediated transient transformation assays (ATTAs) at six optical densities (ODs; 0.05–1.0) with the three most consistent cell death-inducing DsCEs. Whilst DsCEs Ds70057 and Ds131885 triggered strong cell death across all ODs, DsEcp2-1 showed less cell death at lower ODs (Supplementary Fig. S2).

The six DsCEs also differed in their ability to elicit cell death in the non-host plant N. benthamiana. Ds74283 was unique in eliciting consistent and strong cell death in N. benthamiana, despite showing no response in N. tabacum. Only two of the five effectors that induced cell death in N. tabacum (Ds70694 and Ds70057) also consistently induced cell death in N. benthamiana, although it was often not uniform across the infiltrated zone (Fig. 2). Of the other DsCEs, Ds71487 and Ds131885 only induced weak cell death along the perimeter of infiltration zones in less than 50% of infiltrations into N. benthamiana, while DsEcp2-1 caused no cell death (Fig. 2).

To support the premise that the cell death responses were due to cell death elicitation by DsCEs in the apoplast, we cloned them for ATTAs without a secretion signal peptide. As expected, all of the non-secreted versions of these proteins failed to induce cell death (Fig. 2). The lack of cell death was not due to lack of DsCE production in the plant, but rather a lack of DsCE secretion to the apoplast, as each DsCE protein could be detected in infiltrated leaves by western blotting (Supplementary Fig. S3).

Dothistroma septosporum cell death inducers are conserved among fungi

We queried the sequences of each DsCE protein that elicited cell death in Nicotiana spp. to further investigate their similarities to other fungal proteins. Some of these DsCEs are broadly conserved across different fungal classes, and even outside the Ascomycetes (Table 1, Supplementary Table S4). Sequences similar to Ds70057 were the most numerous, with more than half of them found in Eurotiomycete species; at the other extreme, Ds70694 only had homologues in 14 Dothideomycete species. However, both Ds70057 and Ds70694 have multiple paralogues in some species, as well as in the D. septosporum genome, suggesting they are each members of a multi-gene family.

Table 1 BLASTp hits of Dothistroma septosporum cell death elicitors in fungal classes.
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Among the cell-death elicitors, two were notable for their similarity to cell-death inducing proteins that have been described as PAMPs in other fungi. Based on reciprocal BLAST analyses, Ds71487 appears to be an orthologue of RcCDI1 from the barley pathogen Rhynchosporium commune33, with a pairwise identity of 41% and four conserved cysteine residues between the two proteins. Ds131885 is orthologous to VmE02, a cross-kingdom PAMP from the apple pathogen Valsa mali34, with 60% amino acid identity and ten conserved cysteine residues in both proteins.

Infiltration of a DsCE protein into pine shoots induces cell death

Screening candidate effectors for their ability to elicit cell death in model plants is indicative of their potential functions, but screening in a pine host is critical for understanding their actual roles in disease. As a first step to achieving this, we developed a reliable, straight-forward methodology to deliver effector proteins into pine needles (Fig. 3).

Figure 3
figure 3

Overview of the pine shoot vacuum infiltration method developed in this study. Dothistroma septosporum candidate effector Ds70057 and positive control Botrytis cinerea effector BcSSP2 proteins were produced through heterologous expression and secretion by Pichia pastoris (1, 2). The proteins were purified by immobilised metal affinity chromatography (IMAC) (3) then vacuum-infiltrated into clonal microshoots of Pinus radiata (4). Infiltrated shoots were returned to LPch media for up to 7 days after infiltration (5). 1% neutral red was infiltrated to assess the efficiency of vacuum infiltration (6) in the different pine genotypes Sus6 (S6), Sus11 (S11) and Res4 (R4).

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A positive control that elicits cell death when infiltrated into clonal shoots of P. radiata was required to confirm the efficacy of the new method. Consistent with previous studies in which the Botrytis cinerea BcSSP2 effector induced strong cell death in a range of plant species35, vacuum infiltration of purified BcSSP2 protein produced by P. pastoris elicited cell death in three different genotypes of pine shoots within three days, providing an effective positive control.

Importantly, infiltration of the negative controls (protein purification elution buffer or bovine serum albumin protein) did not cause any damage or visible stress responses, as expected, even with higher concentrations of BSA (Fig. 4). In contrast, Pinus radiata shoots infiltrated with purified protein of Ds70057 consistently showed cell death within seven days across all three P. radiata genotypes tested (S6, S11 and R4) (Fig. 4, Supplementary Fig. S4), with these responses near-identical to the positive control BcSSP2.

Figure 4
figure 4

The Dothistroma septosporum candidate effector (DsCE) Ds70057 induces cell death in Pinus radiata shoots following vacuum infiltration. Whole shoots of P. radiata genotypes that are susceptible (S6 and S11) or tolerant (R4) to D. septosporum infection were infiltrated with purified Botrytis cinerea SSP2 (positive control) or DsCE Ds70057 protein produced by heterologous expression in Pichia pastoris. Negative controls were elution buffer only and solutions of bovine serum albumin (BSA). Representative photographs (from 18 to 24 pine shoots for each treatment) were taken 7 days after infiltration, with further images shown in Supplementary Fig. S4. The top panel shows infiltrations of the respective solutions into Nicotiana tabacum (Nt) for reference.

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Discussion

Dothistroma needle blight (DNB) remains a threat to both commercial and native pine forests worldwide, despite decades of research into this disease and its management6,8. Although screening with pathogen effectors based on genome data (effectoromics) has fast-tracked identification of resistant angiosperm crop varieties with corresponding immune receptors, it is not known whether a similar effectoromics-based approach could be applied to improve forest health given the longer life-cycles of forest trees, and the complexity of factors affecting disease resistance36. However, a recent transcriptomics study of Pinus contorta responses to infection with D. septosporum showed up-regulation of pine genes involved in Ca2+ and MAPK defence signalling pathways as well as pathogen-specific defence responses37. Signatures of selection were identified in candidate immune receptors (R genes), suggesting adaptation to the pathogen and the importance of specific interactions between pathogen effectors and host immune receptors in pine defence to D. septosporum.

We built on a prior study38 to identify and refine a small set of secreted proteins that are likely to play a role as effectors in the infection of P. radiata by D. septosporum. A major selection criterion for these proteins was high expression and/or up-regulation during the infection27. We mainly adhered to size and cysteine content thresholds (≤ 300 aa, ≥ 4 Cys) used in studies of angiosperm pathogens39 and used the EffectorP 3.040 tool to help classify the DsCE candidates. The lack of clustering of the 30 DsCE genes on small chromosomes or near repetitive elements (Supplementary Fig. S1, Supplementary Table S1) supports earlier suggestions that, in contrast to several other Dothideomycetes, ‘pathogenesis-associated gene classes’ in D. septosporum were not enriched in these often-hyper-evolving regions41.

Here we used an effectoromics screening approach in two non-hosts to explore the effector arsenal of D. septosporum. Of the 30 small secreted D. septosporum candidate effector (DsCE) proteins that were expressed by transient transformation in N. tabacum or N. benthamiana, six induced cell death. Four of these six proteins are very highly conserved across a broad range of fungal taxa, suggesting some commonality in angiosperm and gymnosperm effectors, as also seen in some Phytophthora pathogens42. Most effectoromics studies include screening on host as well as non-host plants but, to the best of our knowledge, there were no methodologies in place to enable effector protein screening on pines or indeed any other gymnosperms. Thus, we developed a novel method that involves infiltration of purified effector proteins into clonal pine shoots. We validated the system using protein of a highly expressed DsCE, Ds70057, that elicited cell death in N. tabacum, and found that it also triggered cell death across three independent clonal genotypes of P. radiata. Importantly, we showed that P. radiata needles can be screened for responses to potential effectors, suggesting that an effectoromics screen to identify host resistance or susceptibility genes may be possible even in gymnosperm trees.

Functional predictions and screens for homology usually rely on comparisons of primary amino acid sequences using BLASTp. In our study, we also used HHpred to infer possible structural relationships between the 30 DsCEs and proteins with characterised tertiary structures. Notably, HHpred predictions largely supported the BLASTp results, but also uncovered potential functions for five additional DsCEs. One of these five, Ds70694, induced cell death in Nicotiana spp. and was predicted to have structural similarity to proteins with the major human allergen Alt a 1 fold43, with the top HHpred hit to the PAMP-like effector protein PevD1 from Verticillium dahliae44. Another protein with an Alt a1 fold, the Magnaporthe oryzae HR-inducing protein 1 (MoHrip1), is highly induced during the infection of its rice host and has been implicated in promoting disease45. Notably, it has been shown that, like Ds70694, both PevD1 and MoHrip1 can trigger cell death in Nicotiana species44,45,46. This is despite the fact that Nicotiana species are not hosts for D. septosporum, V. dahliae or M. oryzae, suggesting that all three proteins share a conserved function in promoting cell death and/or they possess a conserved epitope that is recognised as a PAMP by these plant species.

Another of the cell-death inducing DsCEs, Ds131885, is an orthologue of VmE02, a cross-kingdom PAMP from the necrotrophic apple pathogen Valsa mali34 that also elicits cell death in Nicotiana spp. VmE02 mutants had reduced numbers of pycnidia, suggesting the protein may be involved in pathogen conidiation. A similar function for Ds131885 would concur with its upregulated expression at the mid and late stages of infection. Recently a receptor-like protein (RLP) in N. benthamiana was identified that mediates VmE02-triggered immunity to fungal and oomycete pathogens47.

Ds71487 is homologous to a Rhynchosporium commune protein, RcCDI1, which induced cell death in N. benthamiana, N. sylvestris, and two other Solanaceae species, but not in its host barley33. RcCDI1 homologues in three other pathogens also induced cell death in N. benthamiana, and the authors concluded that the protein is a PAMP33. Given this context, the induction of cell death in N. tabacum by Ds71487 is not surprising and this response might also be found in other plants. However, it is unclear why this protein only triggered a weak response in N. benthamiana.

The D. septosporum effector protein DsEcp2-1 was already known to elicit cell death in N. tabacum from a previous study25. In that work, DsEcp2-1-deficient mutants showed increased virulence on P. radiata compared to an Ecp2-1-containing wild-type strain, suggesting a possible avirulence role for DsEcp2-1 in eliciting a defence response by the plant. This suggestion supports the concept that D. septosporum effectors could, through adaptation of the host, become a warning signal (avirulence effector) similar to those of biotrophic pathogens48,49.

The remaining DsCEs that caused cell death in non-host plants in our study were not affiliated with any known functions, but were highly conserved across a broad range of fungal taxa (Table 1). Effectors with conserved core functions shared among plant pathogens could be important in elicitation of plant resistance as they are likely to be essential for the microbes’ survival. Mutation or loss of essential conserved pathogen effector genes to escape recognition by a specific plant host immune receptor would most likely lower pathogen viability. It is possible that highly conserved 'core' effectors may have been overrepresented in our results because we screened gymnosperm pathogen effectors in angiosperm non-host plants. However, our results strongly affirm an earlier study that suggests conservation of core effectors between pathogens of angiosperms and gymnosperms42. The matching cell-death responses of pine and Nicotiana sp. to infiltration with Ds70057 and the control B. cinerea SSP2 proteins in our study also suggest similarities in the physiological responses of these disparate plant groups. Similarly, Chen et al.50 found that infiltration of the cerato-platanin protein (CPP) HaCPL2 from Heterobasidion annosum induced cell death in the host Pinus sylvestris when applied to roots in vitro as well as in N. tabacum leaves.

Effectors can sometimes have opposing roles depending on the host context. Whilst they are mainly exploited in effectoromics studies on the basis of a role in avirulence, in which their recognition by a specific plant immune receptor elicits defence, they can have important roles in virulence that may be indispensable for fitness, causing conflicting selection pressures. At the same time, the induction of cell death by the plant host as part of its defence mechanism is beneficial for necrotrophic pathogens, such as Parastagonospora nodorum that causes Septoria blotch of wheat16. The P. nodorum-wheat interaction is regarded as a model pathosystem for understanding the 'inverse gene-for-gene' relationship between effectors and their corresponding plant targets, which are termed susceptibility factors. For both biotrophic and 'inverse' necrotrophic interactions, germplasm can be screened with effector proteins for a resistant or susceptible reaction, respectively, and accordingly selected or deselected for breeding17. However, the situation with hemibiotrophs, such as that of D. septosporum, is more complex and requires accurate understanding of the disease.

The DsCE Ds70057 is of interest due to its very high expression and up-regulation in the mid and late (necrotrophic) stages of disease caused by D. septosporum on P. radiata27 and its ability to elicit cell death in three genotypes of P. radiata as well as in N. tabacum. We suggest it may be a necrotrophic effector triggering cell death that assists the pathogen by eliciting the destruction of needle tissue. This action would augment that of dothistromin which is required for disease lesion expansion, but not for initial elicitation of cell death51. Further studies with Ds70057, including construction and analysis of D. septosporum knockout mutants, will help to determine if Ds70057 is acting as a necrotrophic effector, in which case we would expect these pathogen mutants to have decreased virulence.

An aim of our work was to develop a method to screen putative effectors of conifer pathogens directly in their host using a protein infiltration approach, with the long-term goal of identifying corresponding immune receptors for the effectors, and thus assisting breeding for disease resistance. There were several challenges associated with protein delivery into pines. In an earlier study on P. sylvestris, roots took up purified protein from a root pathogen via filter paper strips, resulting in root cell death50. By submerging pine shoots into solutions containing purified candidate effector proteins, and applying a vacuum pressure, we were able to develop a robust and reliable infiltration process that did not harm the pine needle tissue. We identified a positive control that can be used as a reference in future studies and showed that a secreted protein of D. septosporum induced cell death symptoms in pine shoots as well as Nicotiana leaves.

This novel method of effector delivery to gymnosperm tissue would be well suited for medium-scale screening of multiple pine genotypes with a small number of effector proteins. If the effector-screening process was to be scaled up for high-throughput commercial use with a large number of effector proteins, the time used to grow and prepare fresh purified effector proteins in the P. pastoris system as used here could be a limiting factor. An alternative delivery method might be through Agrobacterium-based transformation of pines, in which the effector genes rather than the proteins are delivered to the host plant. Agrobacterium-based transformation of P. radiata and other Pinus spp. has been achieved52,53, but it is a slow, technically challenging process. Despite this, future efforts to establish reliable transient transformation of pines may be worthwhile.

This study has broad implications for our understanding of fungal pathogens of gymnosperm trees and how these species interact with each other at the molecular level. Importantly, the high level of conservation of effectors across fungal taxa, and conservation of plant responses across angiosperms and gymnosperms suggests that much of what the scientific community has learned about plant-pathogen interactions involving angiosperm systems may also be relevant to gymnosperms. A similar conclusion was reached by studying pine responses to D. septosporum infection37. There also appear to be common 'core' interactions that could be exploited to give broad host-range resistance. Another implication of our work relates to the pine screening method which could be adapted for use with other gymnosperm tree species and other plant hosts that are not amenable to the usual screening methods. Finally, this type of screening method has the potential, along with expression data and pathogen gene knockout studies, to identify necrotrophic effectors that could in turn identify susceptibility gene targets, leading to durable resistance.

Methods

Bacterial strains and plants

Escherichia coli DH5α was used for gene cloning and plasmid propagation. A. tumefaciens GV310154 was used, in conjunction with N. tabacum Wisconsin 38 and N. benthamiana, for ATTA experiments. P. pastoris GS115 was used for heterologous protein expression in liquid culture. P. radiata clonal shoots without roots, grown from embryo cotyledon tissue under sterile conditions on LPch agar55, were used for protein infiltration experiments. P. radiata material used for development of the clonal shoots was obtained with permission from the New Zealand Radiata Pine Breeding Company (RPBC) for the purposes of this research. This study complies with institutional, national and international guidelines and legislation.

DsCE identification and homology searches

Genes encoding conserved candidate secreted apoplastic effector proteins were identified in the D. septosporum genome20, following the bioinformatic pipeline of Hunziker et al.38 in conjunction with transcriptomics data to determine which genes were highly expressed and/or upregulated during infection of P. radiata27. We also used EffectorP 3.040 and comparisons with the characterised apoplastic secretome of C. fulvum22 to generate a short list of 30 predicted apoplastic D. septosporum candidate effector proteins (DsCEs).

BLASTP was used to identify the top homologues of the 30 DsCE proteins in the National Center for Biotechnology Information (NCBI) and Joint Genome Institute (JGI) databases, with an Expected (E)-value threshold of 1E-02. This included a specific query against the foliar pine pathogens Fusarium circinatum (PRJNA565749), Lecanosticta acicola (PRJNA212329), Cronartium ribicola (PRJNA190829), Pseudocercospora pini-densiflorae (PRJNA212512), Cronartium quercuum f. sp. fusiforme (PRJNA67371), Elytroderma deformans (PRJNA537175), Gremmeniella abietina (PRJNA347218) and Cyclaneusma minus (R. McDougal, Scion, unpublished), as well as the pine endophyte Lophodermium nitens (PRJNA335148) (Supplementary Table S3). BLASTp was also used to determine the level of conservation for each of the cell death-eliciting DsCEs across fungal species in JGI, with an E-value threshold of 1E−05. Here, only one isolate per species was examined, with all paralogues from each species retained. Conserved functional domains in the DsCE proteins were identified using the Conserved Domain Database (CDD) in NCBI.

HHpred56 was used to infer possible structural relationships between the 30 DsCEs and proteins of characterised tertiary structure present in the RCSB (Research Collaboratory for Structural Bioinformatics) protein databank. Here, only the top hit was retained. Structural relationships were deemed to be significant if they had a probability score of 95%, an E-value score < 1E−03, and an overall score of > 50. Despite not meeting these criteria, Ds43416 and Ds70694 were also deemed to have significant structural relationships with homologous proteins of C. fulvum through additional support from previous structural predictions22.

Generation of DsCE expression vectors for Agrobacterium tumefaciens-mediated transient transformation assays (ATTA)

DsCE ATTA expression vectors were generated using a method described previously25. Briefly, DsCE genes, without their native signal peptide sequence, were PCR-amplified from D. septosporum NZE10 genomic DNA or cDNA (derived from in vitro or in planta-grown fungus27) using custom primers (Supplementary Table S5; Integrated DNA Technologies) with BsaI recognition sites and overhangs required for modular assembly using the Golden Gate approach57. PCR amplicons were then directly used as entry modules for cloning into the ATTA expression vector pICH86988, which contains a CaMV 35S promoter and octopine synthase terminator flanking BsaI insertion sites58, using Golden Gate assembly, along with an entry module encoding an N-terminal PR1α signal peptide for secretion into the apoplast and an N-terminal 3 X FLAG tag for detection by western blotting. Cell death-eliciting DsCEs were also assembled into expression vectors as above without a PR1α signal peptide. DsCE ATTA expression vectors were then transformed into E. coli, and inserts confirmed by sequencing. Finally, the DsCE ATTA expression vectors, along with the INF1 ATTA expression vector (an extracellular elicitin from Phytophthora infestans59), were transformed into A. tumefaciens by electroporation as described previously25.

Agrobacterium tumefaciens-mediated transient transformation assays (ATTAs)

DsCEs were screened for their ability to elicit cell death in the non-host plants N. tabacum and N. benthamiana using an ATTA, as previously described25,26. Here, single colonies of A. tumefaciens transformed with a DsCE expression vector were first incubated in selective lysogeny broth (LB) at 28 °C overnight. Cells were then collected by centrifugation at 3000\(\times g\) for 5 min, resuspended in infiltration buffer (10 mM MgCl2, 10 mM MES (Sigma-Aldrich, St. Louis, MO, USA) in KOH, pH 5.6, 0.2 mM acetosyringone (Sigma-Aldrich, St. Louis, MO, EUA) to an OD600 of 0.5. Resuspended cultures were incubated at room temperature (RT) for at least 2 h, and infiltrated into the abaxial side of 5–6-week-old N. tabacum and N. benthamiana leaves. At least 12–24 infiltration zones were tested for each treatment (consisting of two leaves from each of two plants × 3–6 repeat experiments). In these ATTAs, INF1 was used as a positive control, while the empty pICH86988 vector was used as a negative control.

To verify the presence of DsCE proteins in agro-infiltrated N. benthamiana leaves, western blotting was carried out as described previously42. Here, total protein extracts from the leaves were separated by SDS-PAGE (12% bis–tris-acrylamide) and transferred onto PVDF membranes. Mouse anti-FLAG(R) antibodies (Sigma Aldrich) and chicken anti-mouse antibodies conjugated with horseradish peroxidase (Santa Cruz Biotechnology, Dallas, TX, USA) were used for detection by an Azure c600 imager (Azure Biosystems, Dublin, CA, USA) after incubation with SuperSignal™ West Dura Extended Duration Signal Substrate (Thermo Fisher Scientific, Waltham, MA, USA).

Development of a protein delivery method for pine tissue

Challenges involved in developing this method included producing DsCE proteins of interest in sufficient purity and quantity, developing an effective protein delivery method, and identifying a positive control. Trials involved in the development of methods to address these challenges were discussed in detail by Hunziker60, and are briefly summarised here.

To produce the DsCE proteins of interest, we first trialled existing ATTA expression constructs and whole N. benthamiana leaves as expression systems for protein production. However, collection of apoplastic wash fluid (AWF), containing secreted DsCEs from infiltrated leaves, did not yield enough protein for replicated studies with pines. We then used heterologous protein expression and secretion in Pichia pastoris, which can be more easily up-scaled. Culture filtrates from P. pastoris transformed with an empty vector (negative control) elicited cell death in some P. radiata genotypes. Thus, to avoid this problematic background response, we purified histidine-tagged DsCEs from culture filtrates of P. pastoris using immobilised metal ion affinity (IMAC).

To deliver purified proteins into pine needles, we trialled several methods. Pine needles were not amenable to syringe infiltration, so vacuum infiltration, with pine tissue immersed in a DsCE protein solution, was used. After infiltration, detached needles, or groups of needles (fascicles), from pine seedlings quickly deteriorated, even when placed in moist conditions, so we chose different plant material for protein delivery assays. Here, clonal shoots of P. radiata produced by tissue culture without roots and maintained on agar medium, were employed55. The shoots were vacuum-infiltrated, then returned to the agar medium for 7‒10 days. Vacuum infiltration of whole shoots with a neutral red dye solution suggested good uptake efficiency.

To identify a positive control, an effector protein from the broad host-range pathogen Botrytis cinerea, BcSSP2, was trialled as it was previously shown to induce strong cell death in N. benthamiana as well as in other plants35.

Heterologous candidate effector protein expression in P. pastoris and protein purification

One of the DsCE proteins, Ds70057, was investigated for its capacity to trigger cell death in pine. Ds70057 was selected because it elicited strong and consistent cell death in N. tabacum, was highly expressed at the 'mid' and 'late' (necrotrophic) stages when lesions are formed by D. septosporum in P. radiata27 and as such was seen as a candidate for playing a role in the switch from biotrophic to necrotrophic growth. For the heterologous expression of Ds70057 in P. pastoris, the cDNA sequence encoding the protein, without its native signal peptide, was PCR-amplified from the previously generated ATTA expression vector. The cDNA sequence for the positive control candidate BcSSP2 was amplified from the plasmid pPICZα-BcSSP235. The primers used are shown in Supplementary Table S5. Resulting PCR amplicons from both candidates were cloned into the expression vector pPic9-His6 (Invitrogen, Carlsberg, CA, USA) behind the α-factor signal peptide sequence using SmaI/EcoRI restriction enzymes and T4 DNA ligase (New England Biolabs, Beverly, MA, USA). Here, the PCR 5′ primer was designed to incorporate a flag-tag for subsequent detection by western blotting. P. pastoris expression vectors were subsequently transformed into E. coli, and their sequence confirmed, as described above. Finally, the expression cassettes were linearised with SacI or SalI restriction enzymes (New England Biolabs) and transformed into P. pastoris according to Kombrink61.

Production of DsCE proteins in liquid culture using P. pastoris was performed according to Weidner et al.62. Expression of candidate proteins was induced by incubation of P. pastoris in 200 mL of BMMY (Buffered Methanol-complex Medium)62 for 72 h, with the successive addition of methanol every 24 h to increase the final concentration by 0.5% (v/v). Following incubation, P. pastoris cells were collected by centrifugation at 4500g for 30 min. The supernatant, containing the secreted protein of interest, was sterilised through a 0.22 µm filter (ReliaPrep, Ahlstrom, Helsinki, Finland) and adjusted to pH 8 through the addition of NaOH.

Secreted candidate proteins were purified by immobilised metal ion affinity (IMAC) using Ni Sepharose 6 Fast Flow (GE Healthcare, Chicago, IL, EUA) according to the manufacturer’s protocol. Before loading, the column (Glass Econo-Column, BioRad, Hercules, CA, EUA) was packed with 5 mL of resin and was 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 and passed through at 1 mL/min. The protein was eluted with an elution buffer (20 mM sodium phosphate, 0.5 M NaCl and 500 mM imidazole, pH 7.4). The elution fractions were mixed, and elution buffer was added to a final volume of 50 mL to obtain enough volume to vacuum-infiltrate pine shoots. A western blot was performed, as above, to determine the presence of the proteins in P. pastoris culture filtrate and after purification.

Pine infiltration with purified proteins

An overview of the pine infiltration method is shown in Fig. 3. Clonal rootless pine shoots were produced by adventitious shoot production from cotyledons55. The shoots were kept in LPch agar55 in glass jars, with each jar containing between six and eight shoots and three jars used for each treatment. For our experiment, two susceptible (S6 and S11) and one resistant (R4) genotype (based on field data) were used. The shoots were completely submerged in approx. 50 mL of each purified DsCE or BcSSP2 protein solution, or in elution buffer or bovine serum albumin (BSA) (Thermo Fisher Scientific) as negative controls63. Samples were exposed to vacuum in a glass chamber for 5 min, prior to gentle release of the vacuum to allow infiltration of the solution. Shoots were then rinsed in sterile MQ water, briefly air-dried, then placed back in the agar medium in the glass jar. The shoots were maintained in a 22 °C room with natural light. Photographs were taken 7 days after infiltration (dai).

Equipment and settings

The heat map in Fig. 1 was generated using heatmap.2 function of the gplots R package64. The photographs in Figs. 2 and 4 were taken using a Nikon D7000 camera using standard automatic exposure settings under a custom-made lighting rig, then cropped as required.