DsEcp2-1 is a polymorphic effector that restricts growth of Dothistroma septosporum in pine
Introduction
Fungal diseases are a persistent threat to plants worldwide. An understanding of how plant-pathogenic 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 cysteine-rich 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), 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 DsEcp2-1) 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 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 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.
Section snippets
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
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 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
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
Conclusions
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
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 Gate cloning vector pICH86988 and Prof. Tancred Frickey (Scion NZ) for orthology searches in the Pinus radiata genome.
References (68)
- et al.
High levels of dothistromin toxin produced by the forest pathogen Dothistroma pini
Mycol. Res.
(2000) - et al.
LaeA negatively regulates dothistromin production in the pine needle pathogen Dothistroma septosporum
Fungal Genet. Biol.
(2016) - et al.
The functional organization of the nopaline A. tumefaciens plasmid pTiC58
Plasmid
(1980) - et al.
A novel GFP-based approach for screening biocontrol microorganisms in vitro against Dothistroma septosporum
J. Microbiol. Meth.
(2011) - et al.
Evolutionary relics dominate the small number of secondary metabolism genes in the hemibiotrophic fungus Dothistroma septosporum
Fungal Biol.
(2019) - et al.
A molecular evolutionary concept connecting nonhost resistance, pathogen host range, and pathogen speciation
Trends Plant Sci.
(2011) - et al.
Natural disulfide bond-disrupted mutants of 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.
(2003) - et al.
Gapped BLAST and PSI-BLAST: a new generation of protein database search programs
Nucl. Acids Res.
(1997) - et al.
Population structure and diversity of an invasive pine needle pathogen reflects anthropogenic activity
Ecol. Evol.
(2014) - et al.
Genome-wide gene expression dynamics of the fungal pathogen Dothistroma septosporum throughout its infection cycle of the gymnosperm host Pinus radiata
Mol. Plant Pathol.
(2016)
A polyketide synthase gene required for biosynthesis of the aflatoxin-like toxin, dothistromin
Mycopathologia
Reduced virulence of an introduced forest pathogen over 50 years
Microorganisms
Global population genomics of the forest pathogen Dothistroma septosporum reveal chromosome duplications in high dothistromin-producing strains
Mol. Plant Pathol.
The veA gene of the pine needle pathogen Dothistroma septosporum regulates sporulation and secondary metabolism
Fungal Genet. Biol.
Understanding plant immunity as a surveillance system to detect invasion
Annu. Rev. Phytopathol.
Conserved fungal LysM effector Ecp6 prevents chitin-triggered immunity in plants
Science
Recognition of Cladosporium fulvum Ecp2 elicitor by non-host Nicotiana spp. is mediated by a single dominant gene that is not homologous to known Cf-genes
Mol. Plant Pathol.
Cladosporium fulvum Effectors: weapons in the arms race with tomato
Annu. Rev. Phytopathol.
Gene for gene models and beyond: the Cladosporium fulvum-tomato pathosystem
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.
Global geographic distribution and host range of Dothistroma species: a comprehensive review
For. Pathol.
A one pot, one step, precision cloning method with high throughput capability
PLoS One.
Interfamily transfer of tomato Ve1 mediates Verticillium resistance in Arabidopsis
Plant Physiol.
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.
Novel mutations detected in avirulence genes overcoming tomato Cf resistance genes in isolates of a Japanese population of Cladosporium fulvum
PLoS One
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.
The biotrophic fungus Cladosporium fulvum circumvents Cf-4-mediated resistance by producing unstable AVR4 elicitors
Plant Cell.
An improved artificial pathogenicity assay for Dothistroma needle blight on Pinus radiata
Australas. Plant Pathol.
Geneious Basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data
Bioinformatics.
Apoplastic recognition of multiple candidate effectors from the wheat pathogen Zymoseptoria tritici in the nonhost plant Nicotiana benthamiana
New Phytol.
A major gene for resistance to white pine blister rust in western white pine from the western cascade range
Phytopathology
Specific HR-associated recognition of secreted proteins from Cladosporium fulvum occurs in both host and non-host plants
Plant J.
The in planta-produced extracellular proteins ECP1 and ECP2 of Cladosporium fulvum are virulence factors
Mol. Plant Microbe Interact.
Recent insights into western white pine genetic resistance to white pine blister rust
Rec. Res. Devel. Biotechnol. Bioeng.
Cited by (9)
Role of pathogen's effectors in understanding host-pathogen interaction
2022, Biochimica et Biophysica Acta - Molecular Cell ResearchCitation Excerpt :This creates an environment conducive to disease transmission by attracting pathogenic microbes to plants. In maize, HC toxin, produced by Cochliobolus carbonum, is a histone deacetylase inhibitor that challenge reconfigures host transcriptional responses, leading to the inability to mount an appropriate immune response [155]. It is imperative to conduct studies before engaging in horizontal gene transfer to ensure that beneficial microbes do not acquire pathogens' toxins.
Dothistroma needle blight
2022, Forest Microbiology: Volume 2: Forest Tree HealthChromosome-level assembly of the Phytophthora agathidicida genome reveals adaptation in effector gene families
2022, Frontiers in Microbiology