Phytophthora species cause many devastating diseases of plants, including those of forest trees. Agathis australis (New Zealand kauri) is an ancient and culturally significant tree species that is susceptible to a lethal root and collar rot caused by Phytophthora agathidicida. Functional characterisation of P. agathidicida virulence factors is limited by difficulties in working with its natural host. The angiosperm Nicotiana benthamiana is widely used as a model plant host for studying plant-pathogen interactions, including P. agathidicida. To help determine whether N. benthamiana is a reliable model for the gymnosperm A. australis, we analysed P. agathidicida gene expression in leaves and roots of N. benthamiana and compared this to an earlier transcriptomic analysis in A. australis. A core set of 1129 genes upregulated in both hosts and organs was enriched for secreted RXLR effector, CAZyme and elicitin proteins, indicating a conserved infection-associated transcriptional programme. Similarities between the suites of genes expressed in leaves and roots support the approach of performing assays of gene function on leaves which are technically easier to use than roots. While many genes were similarly expressed across hosts, there were also some differences. A CAP protein, highly expressed only in N. benthamiana, increased P. agathidicida growth in N. benthamiana but not in A. australis under the conditions tested. These findings suggest that P. agathidicida deploys a largely conserved molecular strategy to colonise gymnosperm and angiosperm hosts, supporting the use of N. benthamiana as a model system for functional studies, while also highlighting pathogen genes that are highly expressed and upregulated in each of the hosts.

Dothistroma needle blight, Cyclaneusma needle blight and red needle cast are devastating foliar pine diseases caused by the fungi Dothistroma septosporum and Cyclaneusma minus and the oomycete Phytophthora pluvialis, respectively. These pathogens colonise the host apoplast, secreting effector proteins to promote infection and disease. If these effectors are recognised by corresponding host resistance proteins, they activate the plant immune system to stop pathogen growth. We aimed to identify and characterise effectors that are common to all three pathogens. Using D. septosporum as a starting point, three candidate effectors (CEs) were investigated: Ds69335 (a CAP protein) and Ds131885, both of which have sequence and structural similarity to CEs of C. minus and P. pluvialis, and Ds74283, which adopts a β-trefoil fold and has structural rather than sequence similarity to CEs from all three pathogens. Notably, of the CEs investigated, Ds74283 and Ds131885, as well as their homologues from C. minus and P. pluvialis, elicited chlorosis or cell death in Nicotiana species, with Ds131885 and its homologues also triggering cell death in Pinus radiata. In line with these observed responses being related to activation of the plant immune system, the chlorosis triggered by Ds131885 and its homologues was compromised in a Nicotiana benthamiana mutant lacking the extracellular immune system co-receptor, SOBIR1. Such cross-kingdom, plant immune system-activating effectors, whether similar in sequence or structure, might ultimately enable the selection or engineering of durable, broad-spectrum resistance against foliar pine pathogens.

Scab or black spot disease, caused by the fungus Venturia inaequalis , is an ongoing threat to commercial apple production. Current control methods involve extensive fungicide use and the deployment of disease-resistant apple cultivars. However, fungicide-resistant strains of V. inaequalis are becoming more prevalent, as are strains that can overcome one or more qualitative disease resistance genes in apple. To increase the durability of disease resistance, and thus decrease our reliance on fungicides, one promising approach could involve stacking endogenous and exogenous resistance genes in apple cultivars using genetic modification. As a starting point for the identification of exogenous resistance genes that are effective against V. inaequalis , 137 candidate effector (CE) proteins from this fungus, fused to a signal peptide for extracellular targeting to the apoplast, were screened for recognition by extracellular leucine-rich repeat (LRR)-receptor-like protein and LRR-receptor-like kinase immune receptors in the model angiosperm species Nicotiana benthamiana and Nicotiana tabacum using Agrobacterium tumefaciens -mediated transient transformation assays. Here, a cell death response in wild-type plants, together with a loss of this response in plants lacking the extracellular immune co-receptor Nb SOBIR1 or Nb BAK1, was used as an indicator of recognition. In total, six CE proteins triggered cell death in one or both Nicotiana species, but only one, a homolog of the Vm E02 effector protein from the apple pathogen Valsa mali , did so in an Nb SOBIR1- and Nb BAK1-dependent manner. The five remaining CE proteins are homologs of other known cell death elicitors from filamentous plant pathogens for which there is evidence that they trigger non-canonical extracellular immunity in plants. One of these is an Alt a 1-like protein that also triggered cell death in apple. Collectively, these findings provide a foundation for the use of a combined set of exogenous and endogenous resistance genes in apple to provide durable protection against scab disease. ### Competing Interest Statement The authors have declared no competing interest.

### Competing Interest Statement The authors have declared no competing interest. Tertiary Education Commission of New Zealand via Bioprotection Aotearoa, 39240 Biotechnology and Biological Sciences Research Council - New Zealand partnering award, BB/T020164/1

Summary Leaf mould, caused by Fulvia fulva, is a devastating disease of tomato plants. In many commercial tomato cultivars, resistance to this disease is governed by the Cf‐9 locus, which encodes five paralogous receptor‐like proteins. Two of these proteins confer resistance: Cf‐9C recognises the previously identified F. fulva effector Avr9 and provides resistance during all plant growth stages, while Cf‐9B recognises the yet‐unidentified F. fulva effector Avr9B and provides mature plant resistance only. In recent years, F. fulva strains have emerged that can overcome the Cf‐9 locus, with Cf‐9C circumvented through Avr9 deletion. To understand how Cf‐9B is circumvented, we set out to identify Avr9B. Comparative genomics, transient expression assays and gene complementation experiments were used to identify Avr9B, while gene sequencing was used to assess Avr9B allelic variation across a world‐wide strain collection. A strict correlation between Avr9 deletion and resistance‐breaking mutations in Avr9B was observed in strains recently collected from Cf‐9 cultivars, whereas Avr9 deletion but no mutations in Avr9B were observed in older strains. This research showcases how F. fulva has evolved to sequentially break down the Cf‐9 locus and stresses the urgent need for commercial tomato cultivars that carry novel, stacked resistance genes active against this pathogen.

Cyclaneusma needle cast, caused by Cyclaneusma minus, affects Pinus species world wide. Previous studies suggested the presence of two distinct morphotypes in New Zealand, ‘verum’ and ‘simile’. Traditional mycological analyses revealed a third morphotype with clear differences in colony morphology and cardinal growth rates at varying temperatures. Genome sequencing of eight C. minus isolates provided further evidence of the existence of a third morphotype, named as ‘novus’ in this study. To further analyse these morphotypes, we predicted candidate effector proteins for all eight isolates, and also characterized a cell-death eliciting effector family, Ecp32, which is present in other pine phytopathogens. In concordance with their distinct classification into three different morphotypes, the number of Ecp32 family members differed, with patterns of pseudogenization in the ‘simile’ morphotype, and some members being found exclusively either in the ‘simile’ or ‘verum’ morphotypes. We also showed that the Ecp32 family proteins trigger cell death in non-host Nicotiana species, and, as previously demonstrated in other plant pathogens, the Ecp32 family proteins in C. minus adopt a β-trefoil fold. These analyses provide further evidence that the three morphotypes might be distinct species that need formal descriptions. Understanding the geographical range of different Cyclaneusma species and variations in virulence and pathogenicity will provide a better understanding of pine needle diseases and enable the development of more durable methods to control this disease.

Phytophthora agathidicida is responsible for a devastating dieback disease that threatens the survival of Agathis australis (kauri), an ancient conifer species endemic to New Zealand. To develop durable control strategies against kauri dieback disease, a better understanding of the host metabolites necessary for the growth and survival of P. agathidicida during in planta growth, particularly during colonisation of the apoplastic environment, where early contact between host and pathogen cells is made, is required. As a starting point to address this knowledge gap, we investigated changes in the metabolite profile of apoplastic washing fluid (AWF) samples harvested from kauri leaves following either mock inoculation or inoculation with P. agathidicida. AWF was extracted from leaves of kauri saplings and inoculated with P. agathidicida on cellophane membranes or cellophane membranes without the pathogen as a control. The metabolite profile of the AWF samples was then analysed via proton nuclear magnetic resonance (1H NMR) spectroscopy at 24 hours and 10 days post-inoculation, and changes investigated relative to the control. Some changes in the metabolite profile of kauri AWF samples following P. agathidicida inoculation were observed using 1H NMR spectroscopy, including a decrease in sucrose and an increase in glucose resulting from the breakdown of more complex carbohydrates. Our results suggest that P. agathidicida modifies or utilises metabolites present in the leaf apoplast of kauri, including carbohydrates that serve as a source of nutrition. These results provide possible new insights into the nutritional requirements of P. agathidicida during apoplastic colonisation of kauri.

Fulvia fulva and Dothistroma septosporum are closely related apoplastic pathogens with similar lifestyles but different hosts: F. fulva is a pathogen of tomato, whilst D. septosporum is a pathogen of pine trees. In 2012, the first genome sequences of these pathogens were published, with F. fulva and D. septosporum having highly fragmented and near-complete assemblies, respectively. Since then, significant advances have been made in unravelling their genome architectures. For instance, the genome of F. fulva has now been assembled into 14 chromosomes, 13 of which have synteny with the 14 chromosomes of D. septosporum, suggesting these pathogens are even more closely related than originally thought. Considerable advances have also been made in the identification and functional characterization of virulence factors (e.g., effector proteins and secondary metabolites) from these pathogens, thereby providing new insights into how they promote host colonization or activate plant defence responses. For example, it has now been established that effector proteins from both F. fulva and D. septosporum interact with cell-surface immune receptors and co-receptors to activate the plant immune system. Progress has also been made in understanding how F. fulva and D. septosporum have evolved with their host plants, whilst intensive research into pandemics of Dothistroma needle blight in the Northern Hemisphere has shed light on the origins, migration, and genetic diversity of the global D. septosporum population. In this review, we specifically summarize advances made in our understanding of the F. fulva-tomato and D. septosporum-pine pathosystems over the last 10 years.

Summary Leaf mould, caused by Fulvia fulva , is a devastating disease of tomato plants. In many commercial tomato cultivars, resistance to this disease is governed by the Cf-9 locus, which comprises five paralogous genes ( Cf-9A–9E ) that encode receptor-like proteins. Two of these proteins contribute to resistance: Cf-9C recognizes the previously identified F. fulva effector Avr9 and provides resistance during all plant growth stages, while Cf-9B recognises the yet-unidentified F. fulva effector Avr9B and provides mature plant resistance only. In recent years, F. fulva strains have emerged that have overcome the Cf-9 locus, with Cf-9C circumvented through Avr9 deletion. To understand how Cf-9B is circumvented, we set out to identify Avr9B . Comparative genomics, in planta transient expression assays and gene complementation experiments were used to identify Avr9B , while gene sequencing was used to assess Avr9B allelic variation across a worldwide strain collection. A strict correlation between Avr9 deletion and resistance-breaking mutations in Avr9B was observed in strains recently collected from Cf-9 cultivars, whereas Avr9 deletion but no mutations in Avr9B were observed in older strains. This research showcases how F. fulva has evolved to sequentially break down the two functional resistance genes of the complex Cf-9 locus and highlights that this locus now has limited value for controlling leaf mould disease in worldwide commercial tomato production.

Plant-pathogenic fungi are a major threat to food security. Among these are subcuticular pathogens, which often cause latent asymptomatic infections, making them difficult to control.

Spray-induced gene silencing (SIGS) involves exogenous spray applications of double-stranded RNA (dsRNA) molecules targeting specific genes, such as those essential to pathogens. This technique has shown great potential for controlling fungal pathogens of horticultural and agricultural plants, but few studies have evaluated its application to forest pathogens. Here we report the first demonstration of exogenous application of dsRNA molecules targeting a gene required for virulence in the foliar pine pathogen Dothistroma septosporum, the causal agent of Dothistroma needle blight (DNB). Constructs expressing sense and antisense dsRNAs were generated targeting a 509 bp fragment of the dothistromin toxin regulatory gene (DsAflR) in D. septosporum. DsAflR is required for the production of dothistromin, a phytotoxin essential for full pathogen virulence. We found that exogenous dsRNA is taken up and the RNA silencing mechanism is functional in D. septosporum through a series of in vitro and in planta trials, such as monitoring the uptake of fluorescently labelled DsAflR-dsRNA into the cell by confocal microscopy and quantifying the extent of gene silencing by quantitative Polymerase Chain Reaction (qPCR). In vitro applications of DsAflR-dsRNA to D. septosporum reduced the accumulation of DsAflR mRNA compared to controls and, in some cases, were found to be statistically significant, despite a high level of variability in the results. Our study also demonstrated a reduction in DNB symptoms on infected pine needles sprayed with DsAflR-dsRNA, which was further supported by a significant reduction in fungal biomass. This preliminary study highlights the potential for SIGS as a future management scheme for controlling the forest pathogen D. septosporum, as well as other forest pathogens.

Biological control of pathogens can be an important tool for long-term management of diseases. Species in the fungal genus Trichoderma have been used for a broad range of agricultural functions including biological control. Here we tested if isolates and mixes of Trichoderma spp. could be effective in reducing symptoms of dothistroma needle blight, caused by the foliar pathogen Dothistroma septosporum, on Pinus radiata. Pot trials with natural and artificial D. septosporum infection and field trials were undertaken. The majority of Trichoderma treatments were not significantly different from the control. However, in one experiment, the Trichoderma treatments significantly reduced disease symptoms in one seedlot but not in the other seedlot tested. Conversely, in the field trial, one Trichoderma treatment significantly increased symptoms in comparison to the control. PCR analysis indicated that Trichoderma can persist over time, 16 months after inoculation, in the soil P. radiata seedlings were grown in, however, further investigation is required. Overall, the results from our trials showed that the Trichoderma isolates and mixes tested were unable to control dothistroma needle blight in P. radiata and are not suitable as biological control agents.

Dothistroma septosporum (Ds) and Fulvia fulva (Ff; previously called Cladosporium fulvum) are two closely related Dothideomycete fungal species that cause Dothistroma needle blight in pine and leaf mold in tomato, respectively. During host colonization, these pathogens secrete virulence factors termed effectors to promote infection. In the presence of corresponding host immune receptors, however, these effectors activate plant defenses, including a localized cell death response that halts pathogen growth. We identified two apoplastic effector protein families, Ecp20 and Ecp32, which are conserved between the two pathogens. The Ecp20 family has four paralogues in both species, while the Ecp32 family has four paralogues in D. septosporum and five in F. fulva. Both families have members that are highly expressed during host infection. Members of the Ecp20 family have predicted structural similarity to proteins with a β-barrel fold, including the Alt a 1 allergen from Alternaria alternata, while members of the Ecp32 family have predicted structural similarity to proteins with a β-trefoil fold, such as trypsin inhibitors and lectins. Using Agrobacterium tumefaciens-mediated transient transformation assays, each family member was assessed for its ability to trigger cell death in leaves of the non-host species Nicotiana benthamiana and N. tabacum. Using this approach, FfEcp20-2, DsEcp20-3, and FfEcp20-3 from the Ecp20 family, and all members from the Ecp32 family, except for the Ds/FfEcp32-4 pair, triggered cell death in both species. This cell death was dependent on secretion of the effectors to the apoplast. In line with recognition by an extracellular immune receptor, cell death triggered by Ds/FfEcp20-3 and FfEcp32-3 was compromised in N. benthamiana silenced for BAK1 or SOBIR1, which encode extracellular co-receptors involved in transducing defense response signals following apoplastic effector recognition. We then investigated whether DsEcp20-3 and DsEcp20-4 triggered cell death in the host species Pinus radiata by directly infiltrating purified protein into pine needles. Strikingly, as in the non-host species, DsEcp20-3 triggered cell death, while DsEcp20-4 did not. Collectively, our study describes two new candidate effector families with cell death-eliciting activity from D. septosporum and F. fulva and provides evidence that members of these families are recognized by plant immune receptors.

Dothistroma needle blight, caused by Dothistroma septosporum, has increased in incidence and severity over the last few decades and is now one of the most important global diseases of pines. Disease resistance breeding could be accelerated by knowledge of pathogen virulence factors and their host targets. However, this is hindered due to inefficient targeted gene disruption in D. septosporum, which is required for virulence gene characterisation. Here we report the first successful application of CRISPR/Cas9 gene editing to a Dothideomycete forest pathogen, D. septosporum. Disruption of the dothistromin pathway regulator gene AflR, with a known phenotype, was performed using nonhomologous end-joining repair with an efficiency of >90%. Transformants with a range of disruption mutations in AflR were produced. Disruption of Ds74283, a D. septosporum gene encoding a secreted cell death elicitor, was also achieved using CRISPR/Cas9, by using a specific donor DNA repair template to aid selection where the phenotype was unknown. In this case, 100% of screened transformants were identified as disruptants. In establishing CRISPR/Cas9 as a tool for gene editing in D. septosporum, our research could fast track the functional characterisation of candidate virulence factors in D. septosporum and helps set the foundation for development of this technology in other forest pathogens.

Ciborinia camelliae (Sclerotiniaceae) is a host- and organ-specific fungal pathogen that causes rapid browning and flower drop on ornamental plants of the genus Camellia. To determine the nature of its necrotrophic factors, we tested whether proteins secreted by C. camelliae can damage host-plant tissues. Fungal culture filtrate caused necrogenic activity, abolished by heat or protease treatments, thus indicating that the secreted necrogenic agents are probably proteinaceous in nature. Mass spectrometry-based proteomics was used to detect and identify secreted proteins of C. camelliae. Proteins secreted in culture media (in vitro) and in petal apoplast (in planta) had similar functional distributions, and key identified proteins included homologs to known virulence factors of the closely related Sclerotiniaceae fungus Botrytis cinerea, including endopolygalacturonases, cerato-platanin family proteins, and necrosis- and ethylene-inducing peptides. The main class of secreted proteins were carbohydrate-active enzymes, a characteristic signature of necrotrophic plant pathogens. Both fungal culture filtrate and apoplastic washes of infected petals induced necrosis when infiltrated into host and nonhost plants. This suggests that while some of the secreted proteins might contribute to virulence of C. camelliae, and can cause necrosis similar to secreted proteins of broad-host Sclerotiniaceae pathogens, they do not have a role in determining its host specificity.

Background Scab, caused by the biotrophic fungus Venturia inaequalis, is the most economically important disease of apples worldwide. During infection, V. inaequalis occupies the subcuticular environment, where it secretes virulence factors, termed effectors, to promote host colonization. Consistent with other plant-pathogenic fungi, many of these effectors are expected to be non-enzymatic proteins, some of which can be recognized by corresponding host resistance proteins to activate plant defences, thus acting as avirulence determinants. To develop durable control strategies against scab, a better understanding of the roles that these effector proteins play in promoting subcuticular growth by V. inaequalis, as well as in activating, suppressing, or circumventing resistance protein-mediated defences in apple, is required. Results We generated the first comprehensive RNA-seq transcriptome of V. inaequalis during colonization of apple. Analysis of this transcriptome revealed five temporal waves of gene expression that peaked during early, mid, or mid-late infection. While the number of genes encoding secreted, non-enzymatic proteinaceous effector candidates (ECs) varied in each wave, most belonged to waves that peaked in expression during mid-late infection. Spectral clustering based on sequence similarity determined that the majority of ECs belonged to expanded protein families. To gain insights into function, the tertiary structures of ECs were predicted using AlphaFold2. Strikingly, despite an absence of sequence similarity, many ECs were predicted to have structural similarity to avirulence proteins from other plant-pathogenic fungi, including members of the MAX, LARS, ToxA and FOLD effector families. In addition, several other ECs, including an EC family with sequence similarity to the AvrLm6 avirulence effector from Leptosphaeria maculans, were predicted to adopt a KP6-like fold. Thus, proteins with a KP6-like fold represent another structural family of effectors shared among plant-pathogenic fungi. Conclusions Our study reveals the transcriptomic profile underpinning subcuticular growth by V. inaequalis and provides an enriched list of ECs that can be investigated for roles in virulence and avirulence. Furthermore, our study supports the idea that numerous sequence-unrelated effectors across plant-pathogenic fungi share common structural folds. In doing so, our study gives weight to the hypothesis that many fungal effectors evolved from ancestral genes through duplication, followed by sequence diversification, to produce sequence-unrelated but structurally similar proteins.

During host colonization, plant-associated microbes, including fungi and oomycetes, deliver a collection of glycoside hydrolases (GHs) to their cell surfaces and surrounding extracellular environments. The number and type of GHs secreted by each organism is typically associated with their lifestyle or mode of nutrient acquisition. Secreted GHs of plant-associated fungi and oomycetes serve a number of different functions, with many of them acting as virulence factors (effectors) to promote microbial host colonization. Specific functions involve, for example, nutrient acquisition, the detoxification of antimicrobial compounds, the manipulation of plant microbiota, and the suppression or prevention of plant immune responses. In contrast, secreted GHs of plant-associated fungi and oomycetes can also activate the plant immune system, either by acting as microbe-associated molecular patterns (MAMPs), or through the release of damage-associated molecular patterns (DAMPs) as a consequence of their enzymatic activity. In this review, we highlight the critical roles that secreted GHs from plant-associated fungi and oomycetes play in plant-microbe interactions, provide an overview of existing knowledge gaps and summarize future directions.

AbstractBackgroundScab, caused by the biotrophic fungusVenturia inaequalis, is the most economically important disease of apples worldwide. During infection,V. inaequalisoccupies the subcuticular environment, where it secretes virulence factors, termed effectors, to promote host colonization. Consistent with other plant-pathogenic fungi, many of these effectors are expected to be non-enzymatic proteins, some of which can be recognized by corresponding host resistance proteins to activate plant defences, thus acting as avirulence determinants. To develop durable control strategies against scab, a better understanding of the roles that these effector proteins play in promoting subcuticular growth byV. inaequalis, as well as in activating, suppressing or circumventing resistance protein-mediated defences in apple, is required.ResultsWe generated the first comprehensive RNA-seq transcriptome ofV. inaequalisduring colonization of apple. Analysis of this transcriptome revealed five temporal waves of gene expression that peaked during early, mid or mid-late infection. While the number of genes encoding secreted, non-enzymatic proteinaceous effector candidates (ECs) varied in each wave, most belonged to waves that peaked in expression during mid-late infection. Spectral clustering based on sequence similarity determined that the majority of ECs belonged to expanded protein families. To gain insights into function, the tertiary structures of ECs were predicted using AlphaFold2. Strikingly, despite an absence of sequence similarity, many ECs were predicted to have structural similarity to avirulence proteins from other plant-pathogenic fungi, including members of the MAX, LARS, ToxA and FOLD effector families. In addition, several other ECs, including an EC family with sequence similarity to the AvrLm6 avirulence effector fromLeptosphaeria maculans, were predicted to adopt a KP6-like fold. Thus, proteins with a KP6-like fold represent another structural family of effectors shared among plant-pathogenic fungi.ConclusionsOur study reveals the transcriptomic profile underpinning subcuticular growth byV. inaequalisand provides an enriched list of ECs that can be investigated for roles in virulence and avirulence. Furthermore, our study supports the idea that numerous sequence-unrelated effectors across plant-pathogenic fungi share common structural folds. In doing so, our study gives weight to the hypothesis that many fungal effectors evolved from ancestral genes through duplication, followed by sequence diversification, to produce sequence-unrelated but structurally similar proteins.

Apple scab, caused by the fungal pathogen Venturia inaequalis, is the most economically important disease of apple (Malus x domestica) worldwide. To develop durable control strategies against this disease, a better understanding of the genetic mechanisms underlying the growth, reproduction, virulence and pathogenicity of V. inaequalis is required. A major bottleneck for the genetic characterization of V. inaequalis is the inability to easily delete or disrupt genes of interest using homologous recombination. Indeed, no gene deletions or disruptions in V. inaequalis have yet been published. Using the melanin biosynthesis pathway gene trihydroxynaphthalene reductase (THN) as a target for inactivation, which has previously been shown to result in a light-brown colony phenotype when transcriptionally silenced using RNA interference, we show, for the first time, that the CRISPR-Cas9 gene editing system can be successfully applied to the apple scab fungus. More specifically, using a CRISPR-Cas9 single guide RNA (sgRNA) targeted to the THN gene, delivered by a single autonomously replicating Golden Gate-compatible plasmid, we were able to identify six of 36 stable transformants with a light-brown phenotype, indicating an ∼16.7% gene inactivation efficiency. Notably, of the six THN mutants, five had an independent mutation. As part of our pipeline, we also report a high-resolution melting (HRM) curve protocol for the rapid detection of CRISPR-Cas9 gene-edited mutants of V. inaequalis. This protocol identified a single base pair deletion mutation in a sample containing only 5% mutant genomic DNA, indicating high sensitivity for mutant screening. In establishing CRISPR-Cas9 as a tool for gene editing in V. inaequalis, we have provided a strong starting point for studies aiming to decipher gene function in this fungus. The associated HRM curve protocol will enable CRISPR-Cas9 transformants to be screened for gene inactivation in a high-throughput and low-cost manner, which will be particularly powerful in cases where the CRISPR-Cas9-mediated gene inactivation efficiency is low.

Phytophthora species are notorious plant pathogens, with some causing devastating tree diseases that threaten the survival of their host species. One such example is Phytophthora agathidicida, the causal agent of kauri dieback - a root and trunk rot disease that kills the ancient, iconic and culturally significant tree species, Agathis australis (New Zealand kauri). A deeper understanding of how Phytophthora pathogens infect their hosts and cause disease is critical for the development of effective treatments. Such an understanding can be gained by interrogating pathogen genomes for effector genes, which are involved in virulence or pathogenicity. Although genome sequencing has become more affordable, the complete assembly of Phytophthora genomes has been problematic, particularly for those with a high abundance of repetitive sequences. Therefore, effector genes located in repetitive regions could be truncated or missed in a fragmented genome assembly. Using a combination of long-read PacBio sequences, chromatin conformation capture (Hi-C) and Illumina short reads, we assembled the P. agathidicida genome into ten complete chromosomes, with a genome size of 57 Mb including 34% repeats. This is the first Phytophthora genome assembled to chromosome level and it reveals a high level of syntenic conservation with the complete genome of Peronospora effusa, the only other completely assembled genome sequence of an oomycete. All P. agathidicida chromosomes have clearly defined centromeres and contain candidate effector genes such as RXLRs and CRNs, but in different proportions, reflecting the presence of gene family clusters. Candidate effector genes are predominantly found in gene-poor, repeat-rich regions of the genome, and in some cases showed a high degree of duplication. Analysis of candidate RXLR effector genes that occur in multicopy gene families indicated half of them were not expressed in planta. Candidate CRN effector gene families showed evidence of transposon-mediated recombination leading to new combinations of protein domains, both within and between chromosomes. Further analysis of this complete genome assembly will help inform new methods of disease control against P. agathidicida and other Phytophthora species, ultimately helping decipher how Phytophthora pathogens have evolved to shape their effector repertoires and how they might adapt in the future.