DsEcp2-1 is a polymorphic effector that restricts growth of Dothistroma septosporum in pine

Journal Pre-proofs DsEcp2-1 is a polymorphic effector that restricts growth of Dothistroma septosporum in pine Yanan Guo, Lukas Hunziker, Carl.H. Mesarich, Pranav Chettri, Pierre-Yves Dupont, Rebecca J. Ganley, Rebecca L. McDougal, Irene Barnes, Rosie E. Bradshaw PII: DOI: Reference:

S1087-1845(19)30203-8 https://doi.org/10.1016/j.fgb.2019.103300 YFGBI 103300

To appear in:

Fungal Genetics and Biology

Received Date: Revised Date: Accepted Date:

30 June 2019 5 November 2019 5 November 2019

Please cite this article as: Guo, Y., Hunziker, L., Mesarich, Carl.H., Chettri, P., Dupont, P-Y., Ganley, R.J., McDougal, R.L., Barnes, I., Bradshaw, R.E., DsEcp2-1 is a polymorphic effector that restricts growth of Dothistroma septosporum in pine, Fungal Genetics and Biology (2019), doi: https://doi.org/10.1016/j.fgb. 2019.103300

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

© 2019 Published by Elsevier Inc.

DsEcp2-1 is a polymorphic effector that restricts growth of Dothistroma septosporum in pine

Yanan Guoa*, Lukas Hunzikera, Carl. H. Mesarichb, Pranav Chettric, Pierre-Yves Dupontd, Rebecca J. Ganleye, Rebecca L. McDougalf, Irene Barnesg and Rosie E. Bradshawa.

aBio-Protection

Research Centre, School of Fundamental Sciences, Massey University,

Palmerston North 4474, New Zealand bBio-Protection

Research Centre, School of Agriculture and Environment, Massey University,

Palmerston North 4474, New Zealand cAgResearch dInstitute eThe

Ltd, Grasslands Research Centre, Palmerston North, New Zealand

of Environmental Science and Research, Christchurch 8041, New Zealand

New Zealand Institute for Plant & Food Research Limited, Te Puke, New Zealand

fScion,

New Zealand Forest Research Institute Ltd, Rotorua 3010, New Zealand

gDepartment

of Genetics, Biochemistry and Microbiology, Forestry and Agricultural

Biotechnology Institute, University of Pretoria, Pretoria, South Africa

* Corresponding author: [email protected]; Tel.: +64-6951-7735

Keywords Dothideomycete; effectors; forest pathogen; immune receptors; plant-pathogen interactions; polymorphisms 1

Abstract The detrimental effect of fungal pathogens on forest trees is an increasingly important problem that has implications for the health of our planet. Despite this, the study of molecular plant-microbe interactions in forest trees is in its infancy, and very little is known about the roles of effector molecules from forest pathogens. Dothistroma septosporum causes a devastating needle blight disease of pines, and intriguingly, is closely related to Cladosporium fulvum, a tomato pathogen in which pioneering effector biology studies have been carried out. Here, we studied D. septosporum effectors that are shared with C. fulvum, by comparing gene sequences from global isolates of D. septosporum and assessing effector function in both host and non-host plants. Many of the effectors were predicted to be non-functional in D. septosporum due to their pseudogenization or low expression in planta, suggesting adaptation to lifestyle and host. Effector sequences were polymorphic among a global collection of D. septosporum isolates, but there was no evidence for positive selection. The DsEcp2-1 effector elicited cell death in the non-host plant Nicotiana tabacum, whilst D. septosporum DsEcp2-1 mutants showed increased colonization of pine needles. Together these results suggest that DsEcp2-1 might be recognized by an immune receptor in both angiosperm and gymnosperm plants. This work may lead to the identification of plant targets for DsEcp2-1 that will provide much needed information on the molecular basis of gymnospermpathogen interactions in forests, and may also lead to novel methods of disease control.

2

1. Introduction Fungal diseases are a persistent threat to plants worldwide. An understanding of how plantpathogenic fungi and their hosts interact at the molecular level is required to inform durable disease resistance breeding programs in plants (Pais et al., 2018). Much of the research carried out on these fungi in recent years has focused on effectors. Effectors are virulence factors that pathogens deploy to promote host colonization, typically by modulating host immune responses (Lo Presti et al., 2015). In the presence of cognate host immune receptors, however, the same effectors instead activate the plant immune system (Cook et al., 2015). This activation renders the fungus avirulent and the plant resistant, and often involves a localized cell death response, termed the hypersensitive response (HR), at the site of invasion (Spoel and Dong, 2012). Molecular interactions between the leaf mold fungus Cladosporium fulvum and its host, tomato, have been studied for decades (de Wit et al., 2009). These interactions serve as a model for studying other pathosystems, particularly those involving Dothideomycete pathogens (de Wit, 2016; Thomma et al., 2005). Many of the well-studied C. fulvum effectors are small secreted cysteinerich proteins delivered to the host apoplast (Mesarich et al., 2018). They include the core effectors Avr4, Ecp2-1 and Ecp6; the term ‘core’ is used to define those effectors that have a conserved biological function to promote the virulence of multiple plant-pathogenic fungi on distantly related host species (Stergiopoulos et al., 2010). Indeed, orthologs of these three effectors have been identified in many other pathogens with diverse hosts, including Zymoseptoria tritici (wheat),

3

Pseudocercospora fijiensis (banana) and Dothistroma septosporum (pine) (de Wit et al., 2012; Marshall et al., 2011; Stergiopoulos et al., 2010). Of the above mentioned core effectors, Avr4 binds to fungal cell wall chitin to protect against degradation by host chitinases (Marshall et al., 2011; van den Burg et al., 2006), whilst Ecp6 sequesters free chitin to prevent recognition by host chitin immune receptors (de Jonge et al., 2010; Sánchez-Vallet et al., 2013). The function of Ecp2-1 remains to be determined (Stergiopoulos et al., 2010). Notably, orthologs of Avr4 and Ecp2-1 from C. fulvum (hereafter referred to as CfAvr4 and CfEcp2-1) in P. fijiensis (MfAvr4 and MfEcp2-1) and D. septosporum (DsAvr4 and DsEcp21) have been found to trigger cell death in plants upon recognition by the cognate tomato immune receptors Cf-4 and CfEcp2, respectively (de Wit et al., 2012; Isaza et al., 2016; Stergiopoulos et al., 2010). This suggests that plants have evolved immune receptors that can mediate recognition of core effectors from different pathogens. Thus, breeding plants with immune receptors that recognize core effectors may provide broad-spectrum resistance. Relatively little is known about the roles that effectors play in the molecular interaction between fungal pathogens and gymnosperm hosts. However, pine (Pinus) trees, like angiosperm plants, are known to possess major immune receptors (Kinloch et al., 1999; Liu et al., 2004; Schoettle et al., 2014; Sniezko et al., 2014), and some gymnosperm pathogens have orthologs of functionally characterized effector genes from angiosperm pathogens in their genomes (Ohm et al., 2012). D. septosporum, a Dothideomycete fungus that contains orthologs of core effectors initially identified in C. fulvum, causes dothistroma needle blight (DNB), a devastating foliar disease of 4

Pinus species (Drenkhan et al., 2016). D. septosporum is very closely related to C. fulvum and its effector genes were predicted as part of a comparative genomics study (de Wit et al., 2012). Further studies showed that D. septosporum DsAvr4 can bind chitin, like CfAvr4 (Mesarich et al., 2015), although studies with CfAvr4 showed that the chitin-binding site and the region recognized by the tomato immune receptor Cf-4 are distinct (Hurlburt et al., 2018). The role of DsAvr4 during infection of pine, and whether pines have a cognate immune receptor for Avr4, is not yet known; a search for orthologs of the tomato immune receptor Cf-4 in the genome of a cultivar of Pinus radiata grown in New Zealand did not reveal any close matches (Personal Communication, Tancred Frickey, Scion NZ). There is an urgent need to develop new tools to combat DNB. Until the 1990s, this disease was mainly a problem in pine plantations in southern hemisphere regions such as Australasia and Africa (Drenkhan et al., 2016). Recent increases in the occurrence and severity of DNB, particularly in the northern hemisphere, have led to major disease epidemics, with changes in climate implicated as a contributing factor (Welsh et al., 2014; Woods et al., 2016). Trees are long-lived, and because there is a constant evolutionary arms race between pathogen effectors and host immune receptors, any efforts to improve the trees’ genetic resistance to pathogens need to consider long-term sustainability (Telford et al., 2015). In this arms race, effectors that have an important role in pathogen virulence are often under positive selection to avoid immune receptor recognition (Sperschneider et al., 2014; Stergiopoulos et al., 2014). In the case of D. septosporum, very little is known about selection and evolution of effector genes. What 5

we know of effectors so far has been deduced from the New Zealand reference genome sequence (de Wit et al., 2012) and there is a need to explore the genetic diversity among D. septosporum isolates from other countries (Barnes et al., 2014). In this work, we addressed the question of whether putative orthologs of C. fulvum effectors might be functional in the D. septosporum–pine interaction and, if so, whether they show evidence of adaptive selection to evade recognition, which might suggest the presence of cognate pine immune receptors. To do this, we investigated allelic variation in a global collection of D. septosporum isolates, assessed their expression and performed functional analyses in model (Nicotiana spp.) and host (Pinus radiata) systems.

2. Material and Methods

2.1 Fungal isolates and culture conditions

A total of 31 D. septosporum isolates, collected over a period of 50 years from 14 different Pinus host species and six geographic regions around the world, were used in this study (Supplementary Table 1). D. septosporum was routinely grown on agar plates made from Dothistroma medium (DM) (Bradshaw et al., 2000) at 22°C. For growth in DM broth, a 4 mm diameter agar plug with D. septosporum mycelium was ground in 100 µl sterile water using a micropestle. Twenty-five µl of ground mycelia were then inoculated into 25 ml of DM broth and 6

incubated at 22°C for 7 days with shaking at 180 rpm. Mycelia to be used for DNA extraction were filtered, snap-frozen in liquid nitrogen and freeze-dried overnight.

2.2 Polymerase chain reaction (PCR) and sequencing

Whole genome sequences, available for 18 D. septosporum isolates from around the world (Bradshaw et al., 2019); (NCBI BioProject accession number PRJNA381823), were screened for homologs of the C. fulvum effectors using tBLASTn (Altschul et al., 1997). In addition, homologous effector gene sequences from 13 additional D. septosporum isolates (Supplementary Table 1) were obtained following PCR amplification. To achieve this, genomic DNA (gDNA) was extracted from freeze-dried mycelium as described (Moller et al., 1992), and the genes amplified from gDNA using PCR primers specific to the 5’ and 3’ flanking regions of the genes (Supplementary Table 2) using platinum Taq DNA high fidelity polymerase (Thermo Fisher Scientific, Waltham, MA, USA) according to manufacturer’s instructions. PCR products were sequenced by Macrogen (South Korea).

2.3 Polymorphism and selection analysis

D.

septosporum

effector

gene

sequences

from

the

New

Zealand

isolate

NZE10

(http://genome.jgi.doe.gov/Dotse1/Dotse1.home.html) were used as references to which the 7

homologous effector gene sequences from all other isolates were compared. Polymorphisms were identified manually based on nucleotide sequence alignments generated using Geneious 9.1.2 (Kearse et al., 2012), and then determined as synonymous or non-synonymous by inspection of amino acid sequence alignments (Supplementary Fig. 1). Maximum likelihood trees were made using Geneious FastTree 2.1.5 (Price et al., 2010). Nucleotide and amino acid sequences for each effector gene and protein, respectively, are shown in Supplementary Table 3. Evidence for genetic selection at individual nucleotide sites of D. septosporum effector genes was assessed using the site models M0 (one ratio), M1 (neutral), M2 (positive selection), M7 (beta), and M8 (beta+ω) in the CODEML program of the PAMLX software package (Xu and Yang, 2013; Yang et al., 2000). A likelihood ratio test (LRT) was used to test whether site models M2 and M8 (testing for positive selection) fit the data significantly (P < 0.05) better than their nested null models M1 and M7 (neutral selection), respectively. Positively selected sites identified using Bayes Empirical Bayes (BEB) and Naive Empirical Bayes (NEB) statistics in both models M2 and M8 were only accepted if the LRT was significant (Yang, 2007).

2.4 Vector construction, targeted gene knockout and complementation

To make targeted gene knockout vectors of the D. septosporum effectors DsAvr4 (pR337), DsEcp2-1 (pR339) and DsEcp6 (pR338), 1 kb of the 3’ and 5’ flanking regions of these genes, and a hygromycin resistance gene-gfp gene fusion (pR408), were amplified by PCR using specific 8

primers carrying attB sites (Supplementary Table 2). The gene knockout vectors were constructed using a Multisite Gateway® three fragment vector construction kit using methods described previously (Chettri et al., 2012). Complementation vectors containing intact copies of the D. septosporum effectors DsAvr4 (pR347), DsEcp2-1 (pR349) and DsEcp6 (pR348) were constructed by restriction/digestion using the restriction endonucleases BamHI and EcoRI (New England Biolabs, Massachusetts, USA) and the DNA ligase T4 (New England Biolabs). The target genes were PCR-amplified as above using primers carrying restriction endonuclease digestion sites (Supplementary Table 2) and cloned into the pBC-phleo vector (pR224) (Silar, 1995). Gene knockout and complementation strains were obtained by protoplast-mediated transformation of the abovementioned pR vectors into D. septosporum NZE10 wild type (WT) and mutant strains, respectively, then confirmed by Southern hybridization, as previously described (Bradshaw et al., 2006) (Supplementary Fig. 2).

2.5 Agrobacterium tumefaciens transient transformation assays

Agrobacterium tumefaciens binary expression vectors for DsEcp2-1, based on pICH86988 (provided by Dr Kee Hoon Sohn, Pohang University of Science and Technology, Korea), were constructed using Golden Gate assembly (Engler et al., 2008). The nucleotide sequence encoding mature DsEcp2-1 (i.e. without a signal peptide) was amplified by PCR from cDNA using primers 9

carrying flanking BsaI sites (Supplementary Table 2). PCR products were purified using a High Pure PCR Product Purification Kit (Roche Life Science, Penzberg, Germany). Following PCR, 10 fmol of purified PCR product were mixed with 10 fmol of DNA encoding an N-terminal PR1α3xFLAG tag (synthesized by Integrated DNA Technologies, Skokie, Illinois USA), as well as 20 fmol pICH86988, in a 20 μl reaction mixture containing 2 μl 0.1% BSA (Thermo Fisher Scientific), 1 μl T4 DNA ligase, 2 μl 10x T4 DNA ligase buffer (Thermo Fisher Scientific) and 1 μl BsaI enzyme (New England Biolabs). Assembly of the final vector was then carried out using a thermocycler with 10 cycles of 37℃ for 5 min and 16℃ for 10 min, 1 cycle of 50℃ for 5 min, and 1 cycle of 80℃ for 5 min. Upon completion, 5 μl of the reaction volume was transformed into Escherichia coli DH5α competent cells by electroporation. The assembled and cloned plasmids were checked by PCR and sequencing. The expression vector for DsAvr4 was constructed previously (Mesarich et al., 2015). Agro-infiltration vectors carrying the correct insert were transformed into A. tumefaciens GV3101 (Holsters et al., 1980). A. tumefaciens transformants were inoculated into 5 ml of lysogeny broth (LB) containing 50 μg/ml of kanamycin, 30 μg/ml of gentamycin and 10 μg/ml of rifampicin, and incubated at 28°C with shaking at 200 rpm overnight. Cultures were pelleted by centrifugation at 4,000 x g for 10 min and resuspended in 5-10 ml infiltration buffer (10 mM MgCl2, 10 mM MES-KOH, pH 5.6, 100 μM acetosyringone) to an OD600 of 1. Resuspended cultures were incubated at room temperature for 3 hours, and infiltrated into the abaxial side of 5 to 6 week old Nicotiana tabacum and N. benthamiana leaves using a 1 ml needleless syringe. Inoculated plants 10

were grown in a greenhouse at 22°C, and at 7 days post-infiltration, were assessed for cell death induction.

2.6 Growth, sporulation and germination phenotype assessments

Radial growth rate was determined as described previously (Chettri and Bradshaw, 2016), except with nine replicates per strain. To assess sporulation, a 50 μl spore suspension (1 x 105 spores/ml) was spread over PMMG (pine extract minimal medium with glucose) agar plates (McDougal et al., 2011), with three replicate plates for each strain. After incubation at 22°C for 7 days, 2 ml of sterile water was added to each plate and kept for 10 min at room temperature. Spores were suspended in sterile water using a sterile glass spreader, and the spore concentrations quantified using a cytometer. To assess germination, 7 day-old spores from PMMG agar plates were suspended in 1 ml Dothistroma sporulation medium (DSM) (Bradshaw et al., 2000) broth and incubated at 22°C for 12 hours with shaking at 150 rpm (n = 3) and the percentage of spores with germ tubes determined.

2.7 Virulence assay and fungal biomass analysis

For the P. radiata infection assay, D. septosporum spores from two independent ∆DsEcp2-1 mutants, one DsEcp2-1-complemented and two WT strains were collected from PMMG agar plates 11

as above. P. radiata trees that were approximately seven months old, and derived from one seed lot, were selected for the D. septosporum infection assay and evenly sprayed with a 3.8 x 106 spores/ml solution. Four replicate trees were used for each of the five D. septosporum strains, plus an uninoculated control, and maintained in four replicate misting chambers as described previously (Kabir et al., 2013). Needles were collected at 11 to 12 weeks post-inoculation and evaluated to determine the percentage of needles with DNB lesions. The fungal biomass of disease lesions was quantified using the single copy D. septosporum DsPksA (polyketide synthase A) gene as target and P. radiata CAD (cinnamyl alcohol dehydrogenase) gene as reference for quantitative real-time PCR as previously described (Chettri et al., 2012). A two-tailed T-test was used to test the hypothesis of no significant difference between WT and mutant or complemented strains at the 95% confidence level.

3. Results

3.1 Many of the Dothistroma septosporum effectors with homology to effectors from Cladosporium fulvum appear to be non-functional

A total of seven effector genes with homology to effector genes from C. fulvum were identified in the D. septosporum NZE10 reference genome. These genes are DsAvr4, DsEcp2-1, DsEcp2-2, DsEcp2-3, DsEcp4, DsEcp5 and DsEcp6. Amongst the seven, DsAvr4, DsEcp2-1, DsEcp2-3, and 12

DsEcp6 are core effector genes (Stergiopoulos et al., 2012; Stergiopoulos et al., 2010). Notably, in a genome comparison (de Wit et al., 2012) and a transcriptomics study from a time course of infected Pinus radiata (Bradshaw et al., 2016), we found earlier that C. fulvum effector homologs present in D. septosporum NZE10 are commonly pseudogenized or expressed at low levels. Of the genes in question, DsEcp2-2, DsEcp4 and DsEcp5 are pseudogenized (de Wit et al., 2012) (Table 1) and, except for DsEcp2-1 and DsEcp6, the in planta expression of the four core effector genes was low, with only 50 or less reads per million per kb (RPMK) (Table 1). The three pseudogenized effector genes (DsEcp2-2, DsEcp4 and DsEcp5) were present in all 18 genomes from a global collection of D. septosporum isolates (Bradshaw et al., 2019) (Supplementary Table 4). We analyzed these sequences to examine the patterns of pseudogenization, and to determine if any of the isolates had functional copies of these genes. DsEcp2-2 contained the same internal stop codon within the signal peptide in all isolates tested (Supplementary Fig. 3, Supplementary Fig. 4 and Supplementary Table 3). For DsEcp4, predicted functional copies were present in both Colombian (COL1 and COL2) isolates. All other isolates (of 31 isolates analyzed) contained the same internal stop codon as the NZE10 isolate, within exon 1, except for the Guatemalan isolates (GUA1 and GUA2), in which a serine was present at the same site. However, both Guatemalan isolates contained a different in-frame stop codon near the start of exon 2, providing evidence for at least two independent pseudogenization events amongst these isolates (Supplementary Fig. 3, Supplementary Fig. 4 and Supplementary Table 3). DsEcp4

13

also had the highest number of nucleotide substitutions (63) amongst the pseudogenes, despite being the shortest gene (Table 1, Supplementary Fig. 3). The third pseudogene, DsEcp5, had an internal stop codon within the signal peptide sequence of all 31 isolates analyzed, except for the Colombian isolates (COL1 and COL2) (Supplementary Fig. 3, Supplementary Fig. 4 and Supplementary Table 3). The COL isolates, however, had a frameshift mutation caused by a single nucleotide deletion in the mature protein coding region, also leading to premature termination of translation (Supplementary Fig. 3). Further evidence for pseudogenization events in DsEcp5 was seen in the Canadian (CAN3) and USA (MON8) isolates that lacked the start codon, as well as in the Guatemalan isolates that had a frameshift mutation due to a nucleotide insertion (Supplementary Fig. 3). The low in planta expression and pseudogenization of these three effector genes, particularly of DsEcp4 and DsEcp5, in which multiple pseudogenization events were evident, suggest ancestral loss of gene function due to selection pressures.

3.2 D. septosporum core effectors are highly polymorphic, but are not under positive selection

Next, we focused on the sequences of the four core effector genes (DsAvr4, DsEcp2-1, DsEcp2-3, and DsEcp6) that are not pseudogenized. Because effectors that have an important role in pathogen virulence are often under positive selection to avoid recognition by immune receptors

14

(Sperschneider et al., 2014; Stergiopoulos et al., 2014), we searched for patterns of polymorphism and evidence of selection in the global isolate collection of D. septosporum. Amongst the four core effectors, some general trends could be seen. Both allelic variation and the numbers of non-synonymous variants were higher in those with low expression (DsEcp2-3 and DsAvr4) compared to those highly expressed in planta (DsEcp2-1 and DsEcp6) (Table 1). This was reflected in the higher numbers of protein variants found amongst the 31 D. septosporum isolates for DsEcp2-3 and DsAvr4 (19 and 13, respectively) than for DsEcp2-1 and DsEcp6 (10 and 6, respectively) (Table 1). Phylogenies of these protein variants (Figure 1) revealed distinct variants, and the highest number of amino acid substitutions, in isolates from Guatemala (GUA) and Colombia (COL) for all four proteins. This was concordant with whole genome studies which showed that GUA and COL isolates belong to a clade distinct from other D. septosporum isolates, including the reference genome isolate NZE10 (Bradshaw et al., 2019). In general, isolates from the southern hemisphere countries South Africa, Kenya, Brazil, Ecuador and Chile showed fewer non-synonymous amino acids compared to those from the northern hemisphere, as might be expected since all comparisons were made to the D. septosporum NZE10 (New Zealand) reference (Fig. 1). Patterns of polymorphism across each of the core effectors were also analyzed (Fig. 2). For DsAvr4, nucleotide polymorphisms that resulted in 19 amino acid substitutions were spread across the protein, but none involved substitution of cysteine residues that are involved in potential

15

disulfide bond formation in DsAvr4. Likewise no amino acid substitutions were present between Cys6 and Cys7, which is known to contain the chitin-binding domain (Mesarich et al., 2015). For DsEcp2-1, the polymorphisms were also spread across the protein. With respect to the cysteine residues, only the region between Cys4 and Cys5 was free from any amino acid substitution, hinting at the possible importance of this region (Fig. 2). Both Colombian (COL) isolates carried an amino acid substitution in the signal peptide (His2 to Gln2), but this was not predicted to affect signal function according to analysis with SignalP 4.0 (Petersen et al., 2011). DsEcp2-3, the core effector with the highest number of DNA polymorphisms, also had an amino acid substitution in the signal peptide that was not predicted to affect its function. Of 38 amino acid substitutions in DsEcp2-3, 21 were only found in the GUA and COL isolates, and over half of these occurred in two regions of the C terminus (Fig. 2). In the first of these C-terminal regions, the two GUA isolates also had a three-nucleotide deletion leading to loss of a serine residue (Ser232) compared to the other isolates. The high variation of amino acid sequence at the C terminus suggests that this part of the protein may be involved in host target interaction. DsEcp6 had the lowest number of amino acid substitutions (six) amongst the core effectors and this was reflected in the conservation of three LysM domains that are associated with chitin binding (de Jonge et al., 2010). The three DsEcp6 LysM motifs showed the highest percentage of amino acid identity (68%, 60% and 77%) to the corresponding CfEcp6 LysM motifs of any other Ecp6 orthologs identified in other fungi so far (Sánchez-Vallet et al., 2013). Of the six nonsynonymous substitutions, five were in the LysM motifs (Fig. 2). Although all four core effectors 16

were polymorphic amongst the D. septosporum isolates tested, there was no evidence for significant levels of positive selection at any sites in any of these genes (LRT >0.05; Supplementary Table 5).

3.3 D. septosporum core effector DsEcp2-1 elicits non-host cell death in N. tabacum

Some pathogen effectors have been shown to induce defense responses in non-host plants, indicative of recognition by non-host immune receptors (Kettles et al., 2017). For example, CfEcp2-1 is able to trigger cell death in several Nicotiana spp. reminiscent of an HR, suggesting recognition by an immune receptor (de Kock et al., 2004; Laugé et al., 2000). To determine if DsEcp2-1 is also able to elicit cell death in Nicotiana species, an Agrobacterium-mediated transient transformation assay was carried out with DsEcp2-1 from NZE10, as well as additional alleles from GUA1 and COL1 isolates that contained high numbers of non-synonymous substitutions (6 and 7, respectively) compared to NZE10 (Fig. 1). DsAvr4 was used as a negative control, as it has previously been shown not to elicit cell death in this system (Mesarich et al., 2015). As expected, DsAvr4 did not elicit cell death in either N. tabacum or N. benthamiana leaves. However, all three DsEcp2-1 isoforms elicited cell death in N. tabacum (Fig. 3), but not in N. benthamiana, as seen with CfEcp2-1 (de Kock et al., 2004).

3.4 D. septosporum ΔDsEcp2-1 mutants showed increased colonization of P. radiata needles 17

The core effectors Avr4, Ecp6 and Ecp2-1 are known virulence factors in C. fulvum (de Jonge et al., 2010; Laugé et al., 1997; van Esse et al., 2007). Therefore, the orthologs of these effectors from D. septosporum were studied in more detail to gain insight into their functions. Targeted gene knockout mutants of DsAvr4 (ΔDsAvr4), DsEcp2-1 (ΔDsEcp2-1) and DsEcp6 (ΔDsEcp6) in D. septosporum NZE10 did not differ in growth rate, sporulation or germination, when compared to the NZE10 WT isolate in culture (Supplementary Table 6). An initial virulence assay (Supplementary Table 7) showed that ΔDsAvr4 and ΔDsEcp6 mutants showed no significant differences in lesion number per needle or lesion size compared to the WT. However, whilst the ΔDsEcp2-1 mutant showed no significant difference in lesion number per needle, it did cause significantly larger lesions, when compared to WT-infected needles (Supplementary Table 7). In other pathosystems, fungal biomass has been used as a parameter for the gain or loss of virulence in planta, e.g. for ΔCfEcp2-1 mutants of C. fulvum (Laugé et al., 1997) and ΔUmTin2 mutants of Ustilago maydis (Tanaka et al., 2014). Thus, the D. septosporum virulence assay was repeated using two independent ΔDsEcp2-1 mutants, DsEcp2-1-complemented and WT strains. Fungal biomass within lesions was estimated using qPCR. The ΔDsEcp2-1 mutants showed 3- to 7-fold more fungal biomass in disease lesions compared to the control strains (Table 2), suggesting that the growth of D. septosporum in disease lesions is restricted in the presence of DsEcp2-1.

4. Discussion 18

4.1 Pseudogenization and low in planta expression of D. septosporum effector genes suggest adaptation to P. radiata

Although D. septosporum is phylogenetically closely related to C. fulvum (de Wit et al., 2012), the two fungi have distantly related hosts, with the former infecting pine (a gymnosperm), and the latter infecting tomato (an angiosperm). The two fungi also have different lifestyles, D. septosporum being a hemibiotroph, and C. fulvum a biotroph. In line with these differences, a comparative genomics study revealed that pseudogenization and gene down-regulation have both played an important role in the adaptation of these two pathogens to their hosts, with secondary metabolite, cell wall degrading enzyme and effector genes most notably affected by these processes (de Wit et al., 2012). These adaptive differences between D. septosporum and C. fulvum were further reinforced by our study. Indeed, all orthologs of known C. fulvum effector genes that we investigated, except for DsEcp6 and DsEcp2-1, showed low levels of expression both in culture and in planta. In other pathosystems, it has been shown that activation of the plant immune system can be circumvented through the down-regulation of a gene encoding an effector that would otherwise be recognized by a corresponding immune receptor. For example, silencing of the Avr3a and Avr1c effector genes from the oomycete pathogen Phytophthora sojae can circumvent Rps3a- and Rps1c-mediated resistance in soybean, respectively (Na et al., 2014; Qutob et al., 2013). It is possible that cognate 19

immune receptors for the lowly expressed D. septosporum effectors are present in P. radiata, and that this low expression enables the pathogen to evade recognition. It is also possible that these effectors are evolutionary relics with no function in D. septosporum, like some of the secondary metabolite genes (Ozturk et al., 2019). Because the only available in planta expression data relate to D. septosporum isolate NZE10, gene expression studies with a wider range of isolates on a wider range of hosts are required to obtain a broader perspective of the biology of these effectors. Three of the D. septosporum effectors investigated in our study, namely DsEcp2-2, DsEcp4 and DsEcp5, were identified as pseudogenes. As with suppression of expression, mutation or loss of an effector through gene deletion can result in the circumvention of resistance mediated by a cognate immune receptor, as has been demonstrated for C. fulvum (Iida et al., 2015; Mesarich et al., 2015). Taken together, these results suggest that DsAvr4, DsEcp2-2, DsEcp2-3, DsEcp4 and DsEcp5 are not essential virulence factors of D. septosporum required for the colonization of P. radiata. However, their pseudogenization or down-regulation may have been selected for in the pathogen population to avoid immune receptor-mediated defense. It is unknown whether the functional isoform of DsEcp4 from the Colombian isolates of D. septosporum could trigger immune receptor-mediated defense in P. radiata.

4.2 The core effectors of D. septosporum are polymorphic, but with no evidence for positive selection

20

A polymorphism analysis provided evidence of diverse effector sequences amongst a global collection of D. septosporum isolates. However, no significant signatures of positive selection at specific sites could be identified. Positive selection was shown for the MfAvr4 and MfEcp2-1 genes of P. fijiensis (Stergiopoulos et al., 2014). However, no reliable evidence of positive selection could be identified in a study of C. fulvum effectors (Stergiopoulos et al., 2007). Despite these apparent differences in selection, mutations have been identified in the Avr4 and Ecp2-1 effectors of C. fulvum and P. fijiensis that can prevent their recognition by the corresponding Cf-4 and Cf-Ecp2 immune receptor proteins in tomato, respectively, using the potato virus X-based transient expression system (Iida et al., 2015; Joosten et al., 1997; Stergiopoulos et al., 2014; van den Burg et al., 2003). As mentioned previously, DsAvr4 is also recognized by the tomato immune receptor protein Cf-4, while DsEcp2-1, like MfEcp2-1, is recognized by the tomato immune receptor Cf-Ecp2 (de Wit et al., 2012; Stergiopoulos et al., 2014). Of the polymorphisms seen in DsAvr4, none were equivalent to the mutations seen in CfAvr4 of C. fulvum that enabled the latter to overcome recognition by Cf-4. C. fulvum isolates that escape Cf-4-mediated defense typically produce an Avr4 isoform with a substitution in one of their cysteine residues. This type of substitution results in the disruption of a disulfide bond, which in turn affects the stability of the protein in the protease-rich environment of the tomato apoplast (Iida et al., 2015; Joosten et al., 1997; van den Burg et al., 2003). For MfEcp2-1 of P. fijiensis, two types of mutations including an insertion (Ser24-Thr25-Gly26-Gln27) or non-synonymous substitutions (Ala60Asn, Ala66His, Asn71Asp and Ala75Ser), have been identified that enable this protein to overcome recognition by 21

Cf-Ecp2 (Stergiopoulos et al., 2014). Even though DsEcp2-1 has amino acid substitutions at equivalent positions to MfEcp2-1 (DsAsn64 (MfAsn60), DsAsp/Glu75 (MfAsp71) and DsSer79 (MfSer75)), these substitutions do not affect recognition by Cf-Ecp2, suggesting that these amino acids are not involved in recognition by Cf-Ecp2.

4.3 DsEcp2-1 triggers cell death in the non-host plant N. tabacum

Agrobacterium tumefaciens transient transformation assays revealed that all three isoforms of DsEcp2-1 identified in our study (from isolates NZE10, COL1 and GUA1) were able to trigger cell death in leaves of the non-host species N. tabacum, but not N. benthamiana. A similar study based on an A. tumefaciens transient transformation assay recently showed that VmHEP1, a homolog of CfEcp2-1 from the necrotrophic fungal pathogen of apple, Valsa mali, can trigger cell death in leaves of N. benthamiana (Zhang et al., 2019), while studies on C. fulvum have shown that CfEcp21 can trigger cell death in leaves of all accessions of N. tabacum, Nicotiana sylvestris and Nicotiana undulata tested, as well all most accessions of Nicotiana paniculata tested, using the PVX-based transient expression system (de Kock et al., 2004; Laugé et al., 2000). Notably, in the study by de Kock et al. (2004), cell death was not elicited by CfEcp2-1 in accessions representing many other species of Nicotiana, including N. benthamiana. Based on the work in N. paniculata, it has been shown that the recognition of CfEcp2-1 is mediated by a yet-unidentified immune receptor encoded

22

by a single dominant gene (Laugé et al., 2000). It is feasible that DsEcp2-1-triggered cell death in N. tabacum is mediated by the same dominant immune receptor-encoding gene. Studies of interactions between effectors and immune receptor proteins have provided important information that can lead to practical solutions for resistance breeding (Nejat et al., 2017; Vleeshouwers and Oliver, 2014). Even though effector recognition in distantly related non-host plants has not frequently been observed (Kettles et al., 2017; Schulze-Lefert and Panstruga, 2011), transfer of a receptor-like protein, Ve1, from tomato to the distantly related species Arabidopsis thaliana provided resistance to Verticillium (Fradin et al., 2011). Thus, it may be possible to transfer an immune receptor gene from N. tabacum to P. radiata, to provide resistance against isolates of D. septosporum carrying DsEcp2-1.

4.4 DsEcp2-1 may be recognized by an immune receptor present in P. radiata

A virulence assay showed that, compared to the WT and ∆DsEcp2-1-complemented strains, the ∆DsEcp2-1 deletion mutants of D. septosporum had significantly increased biomass in disease lesions on P. radiata needles. Hence, the absence of DsEcp2-1 can be linked to increased colonization of the host. This, in turn, could mean that restricted growth in planta of D. septosporum carrying functional DsEcp2-1 is due to recognition of the effector by an immune receptor in P. radiata.

23

Despite the suggestion that DsEcp2-1 has a detrimental effect on growth of D. septosporum when recognized by an immune receptor, the lack of positive selection suggests this effector may also have an important role during infection of pine (i.e. loss of DsEcp2-1 may result in a fitness cost that is greater than the observed decrease in colonization). There are many factors could affect sequence diversification among D. septosporum isolates from different countries and hosts, so it is not trivial to determine what selection pressures might actually be at work within a forest. To provide some insight into potential selection pressures from the P. radiata host, D. septosporum isolates collected over a 50-year period from New Zealand (NZ) forests (four from the 1960s, two from the 1990s and four from the 2000s) were studied (Bradshaw et al submitted). The NZ population of D. septosporum has remained clonal since introduction of the pathogen in the 1960s, in contrast to the high diversity seen in many global populations (Barnes et al., 2014; Bradshaw et al., 2019). Thus the NZ population provides an ideal platform for studying genetic change and adaptation in a forest situation. All seven of the effector genes in the current study, including the in-planta expressed DsEcp2-1 and DsEcp6, had identical nucleotide sequences in all isolates examined. Despite the small sample size, these data suggest that no selection pressure to diversify or lose DsEcp2-1 was imposed in over 50 years in a P. radiata forest. Based on our results, the relevance of DsEcp2-1 for the colonization of pine is not clear. A role for Ecp2-1 proteins in antagonistic microbe–microbe interactions has been proposed, but not yet tested (Stergiopoulos et al., 2010). This is based on the finding that some fungal proteins with an Hce2 (homologs of C. fulvum Ecp2) domain have been identified that also possess a glycoside 24

hydrolase family 18 domain with high similarity to the Zymocin killer toxin from the dairy yeast Kluyveromyces lactis (Stergiopoulos et al., 2012). Another possibility is that Ecp2-1 effectors have different roles in biotrophic and hemibiotrophic fungi (Stergiopoulos et al., 2010). This hypothesis is derived from the finding that the MfEcp2-1 effector from the hemibiotrophic pathogen P. fijiensis is, unlike CfEcp2-1 from the biotroph C. fulvum, able to trigger necrosis in tomato plants lacking the cognate Cf-Ecp2 immune receptor (Stergiopoulos et al., 2010). This necrosis is not as strong as the cell death triggered upon Cf-Ecp2 mediated recognition of Ecp2-1 effectors, suggesting that MfEcp2-1 is able to interact with a plant target (possibly the guardee of Cf-Ecp2 in Cf-Ecp2-carrying plants) to elicit tissue degradation for the transition to necrotrophy (Stergiopoulos et al., 2010). In the presence of the Cf-Ecp2 immune receptor, MfEcp2-1 would be indirectly recognized through its interaction with the virulence target to activate immunity. In further support of the hypothesis, two Ecp2-1 homologs from the necrotrophic fungus Valsa mali (VmHEP1 and VmHEP2) have been found to be required in tandem for full virulence (lesion size) on apple (Zhang et al., 2019). Despite what has been seen in other pathosystems, the mechanism governing the restricted growth of ∆DsEcp2-1 deletion mutants of D. septosporum in pine, and whether DsEcp2-1 also has an important virulence function, remains unclear and requires further investigation.

5. Conclusions

25

This study is one of only a few on the effectors of forest tree pathogens. Here, we studied effectors of the fungal pine needle pathogen D. septosporum that are homologous to well-studied effectors of the closely-related model Dothideomycete C. fulvum. Many of the effectors were predicted to be non-functional in D. septosporum based on their lack of expression in planta or pseudogenization. This is perhaps not surprising, given the different lifestyles and hosts of these pathogens. Of the effectors predicted to be functional, all were polymorphic in sequence among a global set of D. septosporum isolates, but there was no evidence to suggest that positive selection had occurred. The DsEcp2-1 effector elicited a non-host cell death response in N. tabacum, whilst D. septosporum mutants lacking DsEcp2-1 showed increased colonization of pine needles. These results suggest that DsEcp2-1 is recognized by a cognate immune receptor in both angiosperm and gymnosperm plants. Whilst confirmation of the latter is required, these results may lead to identification of host effector targets in pine trees that could inform disease resistance breeding in the future.

Acknowledgements This work was supported by Scion (New Zealand Forest Research Institute; grant number 15325), with assistance from Massey University and the New Zealand Bio-Protection Research Centre. We thank Dr Kee Hoon Sohn (Pohang University of Science and Technology) for providing the Golden

26

Gate cloning vector pICH86988 and Prof. Tancred Frickey (Scion NZ) for orthology searches in the Pinus radiata genome.

Figure legends Fig. 1. Phylogenies of core effector candidates DsAvr4, DsEcp2-1, DsEcp2-3 and DsEcp6 Maximum-likelihood phylogenetic trees of core effectors, constructed by FastTree using amino acid sequences from 31 D. septosporum isolates. The New Zealand isolate (NZE10) was used as the reference to determine the variants. The black arrow and the roman letter (with amino acid changes for each protein variant) indicate the number of protein variants for each effector, compared to the reference NZE10 isolate. BHU1: Bhutan; NZE2, 8 and 10: New Zealand; AUST6: Australia; USA12 and MON8: USA; CAN3: Canada; SAF4 and 1625: South Africa; KEN4: Kenya; GUA1 and 2: Guatemala; COL1 and 2: Colombia; BRZ1: Brazil; CHI17: Chile; ECU3: Ecuador; GRE1: Greece; SLV1: Slovakia; POL4: Poland; CZE1: Czech Republic; AUS4: Austria; HUN253: Hungary; ALP3: Germany; DEN1: Denmark; FRA1: France; SP2: Spain; UK402: England; FIN32: Finland; RUS1: Russia.

Figure 2. Sequence polymorphisms in core effector candidates DsAvr4, DsEcp2-1, DsEcp2-3 and DsEcp6

27

Schematic diagrams showing approximate positions of allelic variations of D. septosporum core effectors DsAvr4, DsEcp2-1, DsEcp2-3 and DsEcp6 using the NZE10 isolate as reference. Dark grey boxes indicate signal peptides, light grey boxes indicate introns and black vertical lines indicate cysteine residues. Horizontal black lines indicate the positions of chitin binding domains (ND, WCDY in DsAvr4) and LysM motifs (in DsEcp6). Flags show positions of nucleotide variations: white are synonymous; black are non-synonymous with the amino acid substitution indicated. Where more than one nucleotide change caused the same amino acid substitution, they are grouped with a square bracket.

Figure 3. Dothistroma septosporum DsEcp2-1 elicits cell death on Nicotiana tabacum. Agrobacterium tumefaciens GV3101 strains carrying D. septosporum effector genes DsAvr4 (NZE10) and DsEcp2-1 variants (NZE10, COL and GUA) were infiltrated into 5 to 6 week old N. tabacum leaves at an OD600 of 1. Phytophthora agathidicida INF1-1, which triggers a hypersensitive response on N. tabacum, was used as positive control and an N-terminus 3xFLAG tagged GFP as negative control. DsAvr4 was unable to elicit cell death whilst all three alleles of DsEcp2-1 did elicit cell death on N. tabacum.

Supplementary Fig. 1 Amino acid sequence alignment of core effectors from 31 Dothistroma septosporum isolates

28

Amino acid alignment of D. septosporum core effectors (A) DsAvr4, (B) DsEcp2-1, (C) DsEcp23 and (D) DsEcp6 from 31 D. septosporum isolates and one C. fulvum isolate. The dark grey boxes show conserved amino acids, the light grey and white boxes show similar amino acids.

Supplementary Fig. 2. Molecular verification of DsAvr4, DsEcp2-1 and DsEcp6 gene replacement and complementation in the D. septosporum genome (A) Schematic diagrams of the restriction enzyme sites, probe binding sites and primers used to confirm ∆DsAvr4, ∆DsEcp2-1 and ∆DsEcp6 knockout (KO) strains of D. septosporum by Southern hybridization and PCR. The blue, black green and red vertical lines show restriction enzyme sites. The horizontal green lines show the probe binding sites. The horizontal blue lines show 5’ and 3’ flanking regions. The large black arrows show DsAvr4, DsEcp2-1 and DsEcp6 coding regions. The large blue arrow shows the hygromycin-gfp replacement cassette in KO strains. The vertical black arrows show the position of primers used to confirm gene complementation in complementation strains. (B) The blue, black, green and red text next to the black arrows (which correspond to the colors used for each restriction enzyme in (A)), and the tables below, show the expected restriction fragment sizes for WT, ∆DsAvr4, ∆DsEcp2-1 and ∆DsEcp6 KO strains in Southern hybridization blots. Both ∆DsAvr4 KO transformants (∆DsAvr4 KO1 and ∆DsAvr4 KO2) lost the WT bands when digested by ScaI or SalI (10557 bp and 7523 bp, respectively) and showed expected ScaI or SalI digestion bands (12221 bp + 977 bp and 1442 bp + 1636 bp, respectively). Similarly for 29

DsEcp2-1, both KO strains (DsEcp2-1 KO1 and DsEcp2-1 KO2) lost the WT bands when digested by ScaI or SalI (8208 bp and 2381 bp, respectively) and showed expected ScaI or SalI digestion bands (4433 bp +6348 bp and 1424 bp + 746 bp, respectively). These results indicate successful targeted replacement of DsAvr4 and DsEcp2-1 with the hygromycin-gfp replacement cassette. Only one DsEcp6 KO strain (DsEcp6 KO1) lost the PstI and HindIII WT fragments (1673 bp and 4733 bp, respectively) and showed correct PstI and HindIII digestion products (3263 bp and 7133 bp, respectively), indicating successful replacement of DsEcp6 with the hygromycin-gfp replacement cassette. The second DsEcp6 KO strain (DsEcp6 KO2) showed both expected WT and KO bands for PstI and HindIII, suggesting that it is an ectopic strain that retains an intact copy of DsEcp6. (C) Confirmation of gene complementation by PCR. Lane L is 1 kb+ ladder. Lane + is the positive control, using NZE10 WT gDNA as template for PCR. The black arrows indicate the size of the expected PCR products. Lanes 1–5 are from PCR screens of five independent complemented transformants for DsAvr4, DsEcp6 and DsEcp2-1. All screened colonies showed bands of the expected size for complementation when screened using gene-specific primers.

Supplementary Fig. 3. Sequence polymorphisms in pseudogenized effector candidates of Dothistroma septosporum Schematic diagrams showing approximate positions of allelic variations of D. septosporum pseudogenes DsEcp2-2, DsEcp4 and DsEcp5 using the NZE10 isolate as reference. Dark grey boxes indicate signal peptides, light grey boxes indicate introns and black vertical lines indicate 30

cysteine residues. Dotted black vertical lines show the positions of internal stop codons for each pseudogene. Flags show positions of nucleotide variations: white are synonymous; black are nonsynonymous with the amino acid substitution indicated. Where more than one nucleotide change caused the same amino acid change, they are grouped with a square bracket.

Supplementary Fig. 4. Phylogenies of pseudogenized effector candidates DsEcp2-2, DsEcp4 and DsEcp5 Approximately-maximum-likelihood phylogenetic trees of pseudogenized effectors constructed by FastTree using amino acid sequences from 31 D. septosporum isolates (except for DsEcp2-2, where 19 D. septosporum isolates were used). The New Zealand isolate (NZE10) was used as the reference to determine the variants. The black arrow and the roman letter (with amino acid changes for each haplotype) indicate the number of haplotypes for each effector. BHU1: Bhutan; NZE2, 8 and 10: New Zealand; AUST6: Australia; USA12 and MON8: USA; CAN3: Canada; SAF4 and 1625: South Africa; KEN4: Kenya; GUA1 and 2: Guatemala; COL1 and 2: Colombia; BRZ1: Brazil; CHI17: Chile; ECU3: Ecuador; GRE1: Greece; SLV1: Slovakia; POL4: Poland; CZE1: Czech Republic; AUS4: Austria; HUN253: Hungary; ALP3: Germany; DEN1: Denmark; FRA1: France; SP2: Spain; UK402: England; FIN32: Finland; RUS1: Russia.

31

References Altschul, S. F., Madden, T. L., Schäffer, A. A., Zhang, J., Zhang, Z., Miller, W., Lipman, D. J., 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389-3402. https://doi.org/10.1093/nar/25.17.3389 Barnes, I., Wingfield, M. J., Carbone, I., Kirisits, T., Wingfield, B. D., 2014. Population structure and diversity of an invasive pine needle pathogen reflects anthropogenic activity. Ecol. Evol. 4, 3642-3661. https://doi.org/doi:10.1002/ece3.1200 Bradshaw, R. E., Ganley, R. J., Jones, W. T., Dyer, P. S., 2000. High levels of dothistromin toxin produced by the forest pathogen Dothistroma pini. Mycol. Res. 104, 325-332. https://doi.org/10.1017/S0953756299001367 Bradshaw, R. E., Guo, Y., Sim, A., Kabir, M. S., Chettri, P., Ozturk, I. K., Hunziker, L., Ganley, R. L., Cox, M. P., 2016. Genome-wide gene expression dynamics of the fungal pathogen Dothistroma septosporum throughout its infection cycle of the gymnosperm host Pinus radiata. Mol. Plant Pathol. 17, 210-224. https://doi.org/10.1111/mpp.12273 Bradshaw, R. E., Jin, H. P., Morgan, B. S., Schwelm, A., Teddy, O. R., Young, C. A., Zhang, S., 2006. A polyketide synthase gene required for biosynthesis of the aflatoxin-like toxin, dothistromin. Mycopathologia. 161, 283-294. https://doi.org/10.1007/s11046-006-0240-5 Bradshaw, R. E., Sim, A. D., Chettri, P., Dupont, P.-Y., Guo, Y., Hunziker, L., McDougal, R. L., Van der Nest, A., Fourie, A., Wheeler, D., Cox, M. P., Barnes, I., 2019. Global population genomics of the forest pathogen Dothistroma septosporum reveal chromosome duplications in high dothistromin-producing strains. Mol. Plant Pathol. 20, 784-799. https://doi.org/10.1111/mpp.12791 Chettri, P., Bradshaw, R. E., 2016. LaeA negatively regulates dothistromin production in the pine needle pathogen Dothistroma septosporum. Fungal Genet. Biol. 97, 24-32. https://doi.org/10.1016/j.fgb.2016.11.001 Chettri, P., Calvo, A. M., Cary, J. W., Dhingra, S., Guo, Y., McDougal, R. L., Bradshaw, R. E., 2012. The veA gene of the pine needle pathogen Dothistroma septosporum regulates sporulation and secondary metabolism. Fungal Genet. Biol. 49, 141-152. https://doi.org/10.1016/j.fgb.2011.11.009 Cook, D. E., Mesarich, C. H., Thomma, B. P. H. J., 2015. Understanding plant immunity as a surveillance system to detect invasion. Annu. Rev. Phytopathol. 53, 541-563. https://doi.org/10.1146/annurev-phyto-080614-120114 de Jonge, R., van Esse, H. P., Kombrink, A., Shinya, T., Desaki, Y., Bours, R., van der Krol, S., Shibuya, N., Joosten, M. H. A. J., Thomma, B. P. H. J., 2010. Conserved fungal LysM effector Ecp6 prevents chitin-triggered immunity in plants. Science 329, 953-955. https://doi.org/10.1126/science.1190859 de Kock, M. J. D., Iskandar, H. M., Brandwagt, B. F., Laugé, R., de Wit, P. J. G. M., Lindhout, P., 2004. Recognition of Cladosporium fulvum Ecp2 elicitor by non-host Nicotiana spp. is 32

mediated by a single dominant gene that is not homologous to known Cf-genes. Mol. Plant Pathol. 5, 397-408. https://doi.org/10.1111/j.1364-3703.2004.00239.x de Wit, P. J. G. M., 2016. Cladosporium fulvum Effectors: weapons in the arms race with tomato. Annu. Rev. Phytopathol. 4, 1-23. https://doi.org/10.1146/annurev-phyto-011516-040249. de Wit, P. J. G. M., Joosten, M. A. J., Thomma, B. P. J., Stergiopoulos, I., Gene for gene models and beyond: the Cladosporium fulvum-tomato pathosystem. In: H. Deising, (Ed.), Plant Relationships. Springer Berlin Heidelberg, 2009, pp. 135-156. de Wit, P. J. G. M., van der Burgt, A., Ökmen, B., Stergiopoulos, I., Abd-Elsalam, K. A., Aerts, A. L., Bahkali, A. H., Beenen, H. G., Chettri, P., Cox, M. P., Datema, E., de Vries, R. P., Dhillon, B., Ganley, A. R., Griffiths, S. A., Guo, Y., Hamelin, R. C., Henrissat, B., Kabir, M. S., Jashni, M. K., Kema, G., Klaubauf, S., Lapidus, A., Levasseur, A., Lindquist, E., Mehrabi, R., Ohm, R. A., Owen, T. J., Salamov, A., Schwelm, A., Schijlen, E., Sun, H., van den Burg, H. A., van Ham, R. C. H. J., Zhang, S., Goodwin, S. B., Grigoriev, I. V., Collemare, J., Bradshaw, R. E., 2012. The genomes of the fungal plant pathogens Cladosporium fulvum and Dothistroma septosporum reveal adaptation to different hosts and lifestyles but also signatures of common ancestry. PLoS Genetics. 8, e1003088. https://doi.org/10.1371/journal.pgen.1003088 Drenkhan, R., Tomešová-Haataja, V., Fraser, S., Bradshaw, R. E., Vahalík, P., Mullett, M. S., Martín-García, J., Bulman, L. S., Wingfield, M. J., Kirisits, T., Cech, T. L., Schmitz, S., Baden, R., Tubby, K., Brown, A., Georgieva, M., Woods, A., Ahumada, R., Jankovský, L., Thomsen, I. M., Adamson, K., Marçais, B., Vuorinen, M., Tsopelas, P., Koltay, A., Halasz, A., La Porta, N., Anselmi, N., Kiesnere, R., Markovskaja, S., Kačergius, A., PapazovaAnakieva, I., Risteski, M., Sotirovski, K., Lazarević, J., Solheim, H., Boroń, P., Bragança, H., Chira, D., Musolin, D. L., Selikhovkin, A. V., Bulgakov, T. S., Keča, N., Karadžić, D., Galovic, V., Pap, P., Markovic, M., Poljakovic Pajnik, L., Vasic, V., Ondrušková, E., Piškur, B., Sadiković, D., Diez, J. J., Solla, A., Millberg, H., Stenlid, J., Angst, A., Queloz, V., Lehtijärvi, A., Doğmuş-Lehtijärvi, H. T., Oskay, F., Davydenko, K., Meshkova, V., Craig, D., Woodward, S., Barnes, I., Cleary, M., 2016. Global geographic distribution and host range of Dothistroma species: a comprehensive review. For. Pathol. 46, 408-442. https://doi.org/10.1111/efp.12290 Engler, C., Kandzia, R., Marillonnet, S., 2008. A one pot, one step, precision cloning method with high throughput capability. PLoS One. 3, e3647. https://doi.org/10.1371/journal.pone.0003647 Fradin, E. F., Abd-El-Haliem, A., Masini, L., van den Berg, G. C. M., Joosten, M. H. A. J., Thomma, B. P. H. J., 2011. Interfamily transfer of tomato Ve1 Mediates Verticillium Resistance in Arabidopsis. Plant Physiol. 156, 2255-2265. https://doi.org/10.1104/pp.111.180067 Holsters, M., Silva, B., van Vliet, F., Genetello, C., de Block, M., Dhaese, P., Depicker, A., Inzé, D., Engler, G., Villarroel, R., van Montagu, M., Schell, J., 1980. The functional

33

organization of the nopaline A. tumefaciens plasmid pTiC58. Plasmid. 3, 212-230. https://doi.org/10.1016/0147-619X(80)90110-9 Hurlburt, N. K., Chen, L. H., Stergiopoulos, I., Fisher, A. J., 2018. Structure of the Cladosporium fulvum Avr4 effector in complex with (GlcNAc)(6) reveals the ligand-binding mechanism and uncouples its intrinsic function from recognition by the Cf-4 resistance protein. PLoS Pathog. 14, e1007263. https://doi.org/10.1371/journal.ppat.1007263 Iida, Y., van't Hof, P., Beenen, H., Mesarich, C., Kubota, M., Stergiopoulos, I., Mehrabi, R., Notsu, A., Fujiwara, K., Bahkali, A., Abd-Elsalam, K., Collemare, J., de Wit, P. J. G. M., 2015. Novel mutations detected in avirulence genes overcoming tomato Cf resistance genes in isolates of a Japanese population of Cladosporium fulvum. PLoS One. 10, e0123271. https://doi.org/10.1371/journal.pone.0123271 Isaza, R. E. A., Diaz-Trujillo, C., Dhillon, B., Aerts, A., Carlier, J., Crane, C. F., de Jong, T. V., de Vries, I., Dietrich, R., Farmer, A. D., 2016. Combating a global threat to a clonal crop: banana black Sigatoka pathogen Pseudocercospora fijiensis (synonym Mycosphaerella fijiensis) genomes reveal clues for disease control. PLoS Genet. 12, e1005876. http://doi:org/10.1371/journal.pgen.1005876 Joosten, M. H. A. J., Vogelsang, R., Cozijnsen, T. J., Verberne, M. C., de Wit, P. J. G. M., 1997. The biotrophic fungus Cladosporium fulvum circumvents Cf-4-mediated resistance by producing unstable AVR4 elicitors. Plant Cell. 9, 367-379. https://doi.org/10.1105/tpc.9.3.367 Kabir, M. S., Ganley, R. J., Bradshaw, R. E., 2013. An improved artificial pathogenicity assay for Dothistroma needle blight on Pinus radiata. Australas. Plant Pathol. 42, 503-510. https://doi.org/10.1007/s13313-013-0217-z Kearse, M., Moir, R., Wilson, A., Stones-Havas, S., Cheung, M., Sturrock, S., Buxton, S., Cooper, A., Markowitz, S., Duran, C., Thierer, T., Ashton, B., Meintjes, P., Drummond, A., 2012. Geneious Basic: An integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics. 28, 1647-1649. https://doi.org/10.1093/bioinformatics/bts199 Kettles, G. J., Bayon, C., Canning, G., Rudd, J. J., Kanyuka, K., 2017. Apoplastic recognition of multiple candidate effectors from the wheat pathogen Zymoseptoria tritici in the nonhost plant Nicotiana benthamiana. New Phytol. 213, 338-350. https://doi.org/10.1111/nph.14215 Kinloch, B. B., Jr., Sniezko, R. A., Barnes, G. D., Greathouse, T. E., 1999. A major gene for resistance to white pine blister rust in western white pine from the western cascade range. Phytopathology. 89, 861-867. https://doi.org/10.1094/phyto.1999.89.10.861 Laugé, R., Goodwin, P. H., de Wit, P. J. G. M., Joosten, M. H. A. J., 2000. Specific HR-associated recognition of secreted proteins from Cladosporium fulvum occurs in both host and nonhost plants. Plant J. 23, 735-745. https://doi.org/10.1046/j.1365-313x.2000.00843.x

34

Laugé, R., Joosten, M., van den Ackerveken, G. F. J. M., van den Broek, H. W. J., de Wit, P. J. G. M., 1997. The in planta-produced extracellular proteins ECP1 and ECP2 of Cladosporium fulvum are virulence factors. Mol. Plant Microbe Interact. 10, 725-734. https://doi.org/10.1094/mpmi.1997.10.6.725 Liu, J. J., Hunt, R. S., Ekramoddoullah, A. K. M., 2004. Recent insights into western white pine genetic resistance to white pine blister rust. Rec. Res. Develop. Biotechnol. Bioeng. 6, 6576. Lo Presti, L., Lanver, D., Schweizer, G., Tanaka, S., Liang, L., Tollot, M., Zuccaro, A., Reissmann, S., Kahmann, R., 2015. Fungal effectors and plant susceptibility. Annu. Rev. Plant Biol. 66, 513-545. https://doi.org/10.1146/annurev-arplant-043014-114623 Marshall, R., Kombrink, A., Motteram, J., Loza-Reyes, E., Lucas, J., Hammond-Kosack, K. E., Thomma, B. P. H. J., Rudd, J. J., 2011. Analysis of two in planta expressed LysM effector homologs from the fungus Mycosphaerella graminicola reveals novel functional properties and varying contributions to virulence on wheat. Plant Physiol. 156, 756-769. https://doi.org/10.1104/pp.111.176347 McDougal, R., Yang, S., Schwelm, A., Stewart, A., Bradshaw, R., 2011. A novel GFP-based approach for screening biocontrol microorganisms in vitro against Dothistroma septosporum. J. Microbiol. Methods. 87, 32-37. https://doi.org/10.1016/j.mimet.2011.07.004 Mesarich, C. H., Okmen, B., Rovenich, H., Griffiths, S. A., Wang, C. C., Jashni, M. K., Mihajlovski, A., Collemare, J., Hunziker, L., Deng, C. H., van der Burgt, A., Beenen, H. G., Templeton, M. D., Bradshaw, R. E., de Wit, P. J. G. M., 2018. Specific hypersensitive responseassociated recognition of new apoplastic effectors from Cladosporium fulvum in wild tomato. Mol. Plant Microbe Interact. 31, 145-162. https://doi.org/10.1094/Mpmi-05-170114-Fi Mesarich, C. H., Stergiopoulos, I., Beenen, H. G., Cordovez, V., Guo, Y., Jashni, M. K., Bradshaw, R. E., de Wit, P. J. G. M., 2015. A conserved proline residue within Dothideomycete Avr4 effector proteins is required to trigger a Cf-4-dependent hypersensitive response. Mol. Plant Pathol. 17, 84-95. https://doi.org/10.1111/mpp.12265 Moller, E. M., Bagnweg, G., Sandermann, H., Geiger, H. H., 1992. A simple and efficient protocol for isolation of high molecular weight DNA from filamentous fungi, fruit body and infected plant tissue. Nucleic Acids Res. 20, 6115-6116. https://doi.org/10.1093/nar/20.22.6115 Na, R., Yu, D., Chapman, B. P., Zhang, Y., Kuflu, K., Austin, R., Qutob, D., Zhao, J., Wang, Y., Gijzen, M., 2014. Genome re-sequencing and functional analysis places the Phytophthora sojae avirulence genes Avr1c and Avr1a in a tandem repeat at a single locus. PLoS One. 9, e89738. https://doi.org/10.1371/journal.pone.0089738 Nejat, N., Rookes, J., Mantri, N. L., Cahill, D. M., 2017. Plant-pathogen interactions: toward development of next-generation disease-resistant plants. Crit. Rev. Biotechnol. 37, 229-237. https://doi.org/10.3109/07388551.2015.1134437 35

Ohm, R. A., Feau, N., Henrissat, B., Schoch, C. L., Horwitz, B. A., Barry, K. W., Condon, B. J., Copeland, A. C., Dhillon, B., Glaser, F., Hesse, C. N., Kosti, I., LaButti, K., Lindquist, E. A., Lucas, S., Salamov, A. A., Bradshaw, R. E., Ciuffetti, L., Hamelin, R. C., Kema, G. H. J., Lawrence, C., Scott, J. A., Spatafora, J. W., Turgeon, B. G., de Wit, P. J. G. M., Zhong, S., Goodwin, S. B., Grigoriev, I. V., 2012. Diverse lifestyles and strategies of plant pathogenesis encoded in the genomes of eighteen Dothideomycetes fungi. PLoS Pathog. 8, e1003037. https://doi.org/10.1371/journal.ppat.1003037 Ozturk, I. K., Dupont, P.-Y., Chettri, P., McDougal, R., Böhl, O. J., Cox, R. J., Bradshaw, R. E., 2019. Evolutionary relics dominate the small number of secondary metabolism genes in the hemibiotrophic fungus Dothistroma septosporum. Fungal Biol. 123, 397-407. https://doi.org/10.1016/j.funbio.2019.02.006 Pais, M., Yoshida, K., Giannakopoulou, A., Pel, M. A., Cano, L. M., Oliva, R. F., Witek, K., Lindqvist-Kreuze, H., Vleeshouwers, V. G. A. A., Kamoun, S., 2018. Gene expression polymorphism underpins evasion of host immunity in an asexual lineage of the Irish potato famine pathogen. BMC Evol. Biol. 18. https://doi.org/10.1186/s12862-018-1201-6 Petersen, T. N., Brunak, S., Von Heijne, G., Nielsen, H., 2011. SignalP 4.0: Discriminating signal peptides from transmembrane regions. Nat. Method. 8, 785-786. https://doi.org/10.1038/nmeth.1701 Price, M. N., Dehal, P. S., Arkin, A. P., 2010. FastTree 2-approximately maximum-likelihood trees for large alignments. PLoS One. 5, e9490. https://doi.org/10.1371/journal.pone.0009490 Qutob, D., Patrick Chapman, B., Gijzen, M., 2013. Transgenerational gene silencing causes gain of virulence in a plant pathogen. Nat. Commun. 4. https://doi.org/10.1038/ncomms2354. Sánchez-Vallet, A., Saleem-Batcha, R., Kombrink, A., Hansen, G., Valkenburg, D. J., Thomma, B., Mesters, J. R., 2013. Fungal effector Ecp6 outcompetes host immune receptor for chitin binding through intrachain LysM dimerization. eLife. 2, e00790. https://doi.org/10.7554/eLife.00790 Schoettle, A. W., Sniezko, R. A., Kegley, A., Burns, K. S., 2014. White pine blister rust resistance in limber pine: evidence for a major gene. Phytopathology. 104, 163-173. https://doi.org/10.1094/PHYTO-04-13-0092-R. Schulze-Lefert, P., Panstruga, R., 2011. A molecular evolutionary concept connecting nonhost resistance, pathogen host range, and pathogen speciation. Trends Plant Sci. 16, 117-125. https://doi.org/10.1016/j.tplants.2011.01.001. Silar, P., 1995. Two new easy to use vectors for transformations. Fungal Genet. Newsl. 42. https://doi.org/10.4148/1941-4765.1353 Sniezko, R. A., Smith, J., Liu, J.-J., Hamelin, R. C., 2014. Genetic resistance to fusiform rust in southern pines and white pine blister rust in white pines - A contrasting tale of two rust pathosystems - current status and future prospects. Forests. 5, 2050-2083. https://doi.org/10.3390/f5092050

36

Sperschneider, J., Ying, H., Dodds, P. N., Gardiner, D. M., Upadhyaya, N. M., Singh, K. B., Manners, J. M., Taylor, J. M., 2014. Diversifying selection in the wheat stem rust fungus acts predominantly on pathogen-associated gene families and reveals candidate effectors. Front. Plant Sci. 5. https://doi.org/10.3389/fpls.2014.00372 Spoel, S. H., Dong, X., 2012. How do plants achieve immunity? Defence without specialized immune cells. Nat. Rev. Immunol. 12, 89-100. https://doi.org/10.1038/nri3141. Stergiopoulos, I., Cordovez, V., Ökmen, B., Beenen, H. G., Kema, G. H. J., de Wit, P. J. G. M., 2014. Positive selection and intragenic recombination contribute to high allelic diversity in effector genes of Mycosphaerella fijiensis, causal agent of the black leaf streak disease of banana. Mol. Plant Pathol. 15, 447-460. https://doi.org/10.1111/mpp.12104 Stergiopoulos, I., de Kock, M. J. D., Lindhout, P., de Wit, P. J. G. M., 2007. Allelic variation in the effector genes of the tomato pathogen Cladosporium fulvum reveals different modes of adaptive evolution. Mol. Plant Microbe Interact. 20, 1271-1283. https://doi.org/10.1094/mpmi-20-10-1271 Stergiopoulos, I., Kourmpetis, Y. A. I., Slot, J. C., Bakker, F. T., de Wit, P. J. G. M., Rokas, A., 2012. In silico characterization and molecular evolutionary analysis of a novel superfamily of fungal effector proteins. Mol. Biol. Evol. 29, 3371-3384. https://doi.org/10.1093/molbev/mss143 Stergiopoulos, I., van den Burg, H. A., Okmen, B., Beenen, H. G., van Liere, S., Kema, G. H. J., de Wit, P. J. G. M., 2010. Tomato Cf resistance proteins mediate recognition of cognate homologous effectors from fungi pathogenic on dicots and monocots. Proc. Natl. Acad. Sci. U.S.A. . 107, 7610-7615. https://doi.org/10.1073/pnas.1002910107 Tanaka, S., Brefort, T., Neidig, N., Djamei, A., Kahnt, J., Vermerris, W., Koenig, S., Feussner, K., Feussner, I., Kahmann, R., 2014. A secreted Ustilago maydis effector promotes virulence by targeting anthocyanin biosynthesis in maize. eLife. 3, e01355. https://doi.org/10.7554/eLife.01355 Telford, A., Cavers, S., Ennos, R. A., Cottrell, J. E., 2015. Can we protect forests by harnessing variation in resistance to pests and pathogens? Forestry. 88, 3-12. https://doi.org/10.1093/forestry/cpu012 Thomma, B. P. G. M., Van Esse, H. P., Crous, P. W., de Wit, P. J. G. M., 2005. Cladosporium fulvum (syn. Passalora fulva), a highly specialized plant pathogen as a model for functional studies on plant pathogenic Mycosphaerellaceae. Mol. Plant Pathol. 6, 379-393. https://doi.org/10.1111/j.1364-3703.2005.00292.x van den Burg, H. A., Harrison, S. J., Joosten, M. H. A. J., Vervoort, J., de Wit, P. J. G. M., 2006. Cladosporium fulvum Avr4 protects fungal cell walls against hydrolysis by plant chitinases accumulating during infection. Mol. Plant Microbe Interact. 19, 1420-1430. https://doi.org/10.1094/mpmi-19-1420 van den Burg, H. A., Westerink, N., Francoijs, K. J., Roth, R., Woestenenk, E., Boeren, S., de Wit, P. J. G. M., Joosten, M., Vervoort, J., 2003. Natural disulfide bond-disrupted mutants of 37

AVR4 of the tomato pathogen Cladosporium fulvum are sensitive to proteolysis, circumvent Cf-4-mediated resistance, but retain their chitin binding ability. J. Biol. Chem. 278, 27340-27346. https://doi.org/10.1074/jbc.M212196200 van Esse, H. P., Bolton, M. D., Stergiopoulos, L., de Wit, P. J. G. M., Thomma, B. P. H. J., 2007. The chitin-binding Cladosporium fulvum effector protein Avr4 is a virulence factor. Mol. Plant Microbe Interact. 20, 1092-1101. https://doi.org/10.1094/mpmi-20-9-1092 Vleeshouwers, V. G. A. A., Oliver, R. P., 2014. Effectors as tools in disease resistance breeding against biotrophic, hemibiotrophic, and necrotrophic plant pathogens. Mol. Plant Microbe Interact. 27, 196-206. https://doi.org/10.1094/mpmi-10-13-0313-ia Welsh, C., Lewis, K. J., Woods, A. J., 2014. Regional outbreak dynamics of Dothistroma needle blight linked to weather patterns in British Columbia, Canada. Can. J. For. Res. 44, 212219. https://doi.org/10.1139/x92-147 Woods, A. J., Martín-García, J., Bulman, L., Vasconcelos, M. W., Boberg, J., La Porta, N., Peredo, H., Vergara, G., Ahumada, R., Brown, A., Diez, J. J., 2016. Dothistroma needle blight, weather and possible climatic triggers for the disease's recent emergence. For. Pathol. 46, 443-452. https://doi.org/10.1111/efp.12248 Xu, B., Yang, Z. H., 2013. pamlX: A graphical user interface for PAML. Mol. Biol. Evol. 30, 27232724. https://doi.org/10.1093/molbev/mst179 Yang, Z., 2007. PAML 4: phylogenetic analysis by maximum likelihood. Mol. Biol. Evol. 24, 1586-1591. https://doi.org/10.1093/molbev/msm088 Yang, Z., Nielsen, R., Goldman, N., Pedersen, A. M., 2000. Codon-substitution models for heterogeneous selection pressure at amino acid sites. Genetics 155, 431-449. Zhang, M., Xie, S., Zhao, Y., Meng, X., Song, L., Feng, H., Huang, L., 2019. Hce2 domaincontaining effectors contribute to the full virulence of Valsa mali in a redundant manner. Mol. Plant Pathol. 20, 843-856. https://doi.org/doi:10.1111/mpp.12796

38

Highlights 

Dothistroma septosporum (Ds) and Cladosporium fulvum share effector genes



In Ds, many of these are pseudogenized or are poorly expressed in planta



All these effectors are polymorphic among global Ds isolates



DsEcp2-1 triggers cell death in a non-host plant and restricts Ds growth in pine



These results suggest both pathogen and host adaptation at the molecular level

39

Table 1. Dothistroma septosporum effectors with homologs in Cladosporium fulvum Effector genea

JGI protein ID

DsAvr4

36707

DsEcp2-1

158381

DsEcp2-2a 23431 DsEcp2-3

127671

DsEcp4a

192200

DsEcp5a

69490

DsEcp6

46236

a DsEcp2-2,

Size Amino acid (nt) 147 (498) 168 (576) 148 (739) 311 (995) 124 (606) 148 (413) 228 (444)

Cysteine Best BLASTp hitb (eresidues value, % aa identity)

Total nt changes (nonsynonymous)

8

31 (19)

Numb protei varian 13

28 (12)

10

41 (21)

8

76 (38)

19

63 (37)

10

26 (17)

9

27 (6)

6

Cf Avr4 JGI189855 (8.0e35, 59%) Pf hypothetical protein JGI52972 (1.6e-51, 67%)d Cf hypothetical protein JGI193474 (2.7e-33, 49%) Cf Ecp2-3 JGI195482 (2.7e102, 56%) Cf Ecp4 JGI186834 (5.9e36, 56%) Cf Ecp5 GenBank CAC01610.1 (2.0e-19, 39%) Cf Ecp6 JGI189398 (1.1e99, 64%)

5 2 5 6 7 9

DsEcp4 and DsEcp5 are pseudogenes.

b

Cf: Cladosporium fulvum; Pf: Pseudocercospora fijiensis.

c

Gene expression in Reads Per Million per Kilobase (RPMK) from Bradshaw et al. (2016) with

early, mid and late representing stages of infection in planta. d

DsEcp2-1 is the only effector listed here that shows a higher amino acid identity to P. fijiensis

than C. fulvum.

40

Table 2. Effects of DsEcp2-1 deletion on Dothistroma septosporum biomass in Pinus radiata needle lesions

Relative fungal

Straina

Fungal DNA (ng

biomassb

per mg dry sample)d

Fungal DNA (ng per lesion)

ne

Wild-type

0.05 ± 0.01

4.45 ± 0.63

4.45 ± 0.63

4

ΔDsEcp2-1A

0.12 ± 0.02*

14.31 ± 1.18*

14.31 ± 1.18*

4

ΔDsEcp2-1B

0.34 ± 0.11*

36.60 ± 7.68*

36.60 ± 7.68*

4

CO-DsEcp2-1

0.05 ± 0.01

4.28 ± 0.87

4.28 ± 0.87

3

aTwo

independent knockout mutants of DsEcp2-1 (A and B) and a mutant complemented with the

DsEcp2-1 gene (CO). bFungal

biomass relative to pine, determined by CV values from qPCR of fungal DsPksA gene

target/pine CAD reference (mean ± SD). An asterisk indicates significant difference (P < 0.05) between the values for each mutant, or complemented mutant, compared to the wild type. cFungal

DNA (ng) per mg dry weight of infected needle lesion sample (mean ± SD).

dFungal

DNA (ng) per lesion (mean ± SD).

eNumber

of P. radiata plants inoculated per strain.

41

42

43

44