Sequential breakdown of the Cf-9 leaf mould resistance locus in tomato by Fulvia fulva.

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.

The Bcvic1 and Bcvic2 vegetative incompatibility genes in Botrytis cinerea encode proteins with domain architectures involved in allorecognition in other filamentous fungi.

Vegetative incompatibility is a fungal allorecognition system characterised by the inability of genetically distinct conspecific fungal strains to form a viable heterokaryon and is controlled by multiple polymorphic loci termed vic (vegetative incompatibility) or het (heterokaryon incompatibility). We have genetically identified and characterised the first vic locus in the economically important, plant-pathogenic, necrotrophic fungus Botrytis cinerea. A bulked segregant approach coupled with whole genome Illumina sequencing of near-isogenic lines of B. cinerea was used to map a vic locus to a 60-kb region of the genome. Within that locus, we identified two adjacent, highly polymorphic open reading frames, Bcvic1 and Bcvic2, which encode predicted proteins that contain domain architectures implicated in vegetative incompatibility in other filamentous fungi. Bcvic1 encodes a predicted protein containing a putative serine esterase domain, a NACHT family of NTPases domain, and several Ankyrin repeats. Bcvic2 encodes a putative syntaxin protein containing a SNARE domain; such proteins typically function in vesicular transport. Deletion of Bcvic1 and Bcvic2 individually had no effect on vegetative incompatibility. However, deletion of the region containing both Bcvic1 and Bcvic2 resulted in mutant lines that were severely restricted in growth and showed loss of vegetative incompatibility. Complementation of these mutants by ectopic expression restored the growth and vegetative incompatibility phenotype, indicating that Bcvic1 and Bcvic2 are controlling vegetative incompatibility at this vic locus.

Cell Wall Carbohydrate Dynamics during the Differentiation of Infection Structures by the Apple Scab Fungus, Venturia inaequalis.

Scab, caused by the biotrophic fungal pathogen Venturia inaequalis, is the most economically important disease of apples. During infection, V. inaequalis colonizes the subcuticular host environment, where it develops specialized infection structures called runner hyphae and stromata. These structures are thought to be involved in nutrient acquisition and effector (virulence factor) delivery, but also give rise to conidia that further the infection cycle. Despite their importance, very little is known about how these structures are differentiated. Likewise, nothing is known about how these structures are protected from host defenses or recognition by the host immune system. To better understand these processes, we first performed a glycosidic linkage analysis of sporulating tubular hyphae from V. inaequalis developed in culture. This analysis revealed that the V. inaequalis cell wall is mostly composed of glucans (44%) and mannans (37%), whereas chitin represents a much smaller proportion (4%). Next, we used transcriptomics and confocal laser scanning microscopy to provide insights into the cell wall carbohydrate composition of runner hyphae and stromata. These analyses revealed that, during subcuticular host colonization, genes of V. inaequalis putatively associated with the biosynthesis of immunogenic carbohydrates, such as chitin and ß-1,6-glucan, are downregulated relative to growth in culture, while on the surface of runner hyphae and stromata, chitin is deacetylated to the less-immunogenic carbohydrate chitosan. These changes are anticipated to enable the subcuticular differentiation of runner hyphae and stromata by V. inaequalis, as well as to protect these structures from host defenses and recognition by the host immune system. IMPORTANCE 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. A key feature of these pathogens is their ability to differentiate specialized subcuticular infection structures that, to date, remain largely understudied. This is typified by Venturia inaequalis, which causes scab, the most economically important disease of apples. In this study, we show that, during subcuticular host colonization, V. inaequalis downregulates genes associated with the biosynthesis of two immunogenic cell wall carbohydrates, chitin and ß-1,6-glucan, and coats its subcuticular infection structures with a less-immunogenic carbohydrate, chitosan. These changes are anticipated to enable host colonization by V. inaequalis and provide a foundation for understanding subcuticular host colonization by other plant-pathogenic fungi. Such an understanding is important, as it may inform the development of novel control strategies against subcuticular plant-pathogenic fungi.

Biotechnological approaches for predicting and controlling apple storage disorders.

Fruit storage disorders are major causes of crop losses and downgrades. Cold storage, either in air or in controlled atmospheres high in CO2 and low in O2, can result in chilling injury or respiratory injury (due to high internal CO2 concentrations). Here, we review biotechnological approaches currently being used to better understand these processes, to predict to provide resistance/tolerance to them. Reducing postharvest crop losses through improved cultivars or inventory management will be a major contributor to food security.

The Venturia inaequalis effector repertoire is dominated by expanded families with predicted structural similarity, but unrelated sequence, to avirulence proteins from other plant-pathogenic fungi.

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.

Biotechnological approaches for reducing fruit losses caused by pathogenic infection.

Fruit loss due to disease occurs in both the field and postharvest. Knowledge of host immune responses and pathogen virulence is enabling the formulation of increasingly sophisticated strategies for disease control. Traditional genetic modification, typically involving overexpression of genes involved in pathogen perception and defence responses, is beginning to be superseded by CRISPR-Cas9 manipulation of host susceptibility targets. Moreover, the refinement of RNA interference (RNAi) strategies, including spray-induced gene silencing (SIGS), is allowing more nuanced control options. These latter approaches have the advantage over earlier technologies in that either they do not result in the generation of genetically modified organisms (RNAi-based SIGS), or the genetic manipulation used leaves no trace of introduced genetic material (gene editing). Thus, these strategies may be more widely acceptable for deployment for future disease control.

Biotechnological approaches for controlling postharvest fruit softening.

Fruit softening is the major factor determining the postharvest life of fruit, affecting bruise and damage susceptibility, pathogen colonisation, and consumer satisfaction, all of which contribute to product losses in the supply chain and consumers' homes. Ripening-related changes to the cell wall, cuticle and soluble sugars largely determine softening, and some are amenable to biotechnological intervention, for example, by manipulation of the expression of genes encoding cell wall-modifying proteins or wax and cutin synthases. In this review, we discuss work exploring the role of genes involved in cell wall and cuticle properties, and recent developments in the silencing of multiple genes by targeting single transcription factors. Identification of transcription factors that control the expression of suites of genes encoding cell wall-modifying proteins provides exciting targets for biotechnology.

CRISPR-Cas9 gene editing and rapid detection of gene-edited mutants using high-resolution melting in the apple scab fungus, Venturia inaequalis.

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.

Search for host defense markers uncovers an apple agglutination factor corresponding with fire blight resistance.

Pathenogenesis-related (PR) proteins are extensively used as molecular markers to dissect the signaling cascades leading to plant defense responses. However, studies focusing on the biochemical or biological properties of these proteins remain rare. Here, we identify and characterize a class of apple (Malus domestica) PR proteins, named M. domestica AGGLUTININS (MdAGGs), belonging to the amaranthin-like lectin family. By combining molecular and biochemical approaches, we show that abundant production of MdAGGs in leaf tissues corresponds with enhanced resistance to the bacterium Erwinia amylovora, the causal agent of the disease fire blight. We also show that E. amylovora represses the expression of MdAGG genes by injecting the type 3 effector DspA/E into host cells and by secreting bacterial exopolysaccharides. Using a purified recombinant MdAGG, we show that the protein agglutinates E. amylovora cells in vitro and binds bacterial lipopolysaccharides at low pH, conditions reminiscent of the intercellular pH occurring in planta upon E. amylovora infection. We finally provide evidence that negatively charged polysaccharides, such as the free exopolysaccharide amylovoran progressively released by the bacteria, act as decoys relying on charge-charge interaction with the MdAGG to inhibit agglutination. Overall, our results suggest that the production of this particular class of PR proteins may contribute to apple innate immunity mechanisms active against E. amylovora.

Reference genes for gene expression analysis in the fungal pathogen Neonectria ditissima and their use demonstrating expression up-regulation of candidate virulence genes.

European canker, caused by the necrotrophic fungal phytopathogen Neonectria ditissima, is one of the most damaging apple diseases worldwide. An understanding of the molecular basis of N. ditissima virulence is currently lacking. Identification of genes with an up-regulation of expression during infection, which are therefore probably involved in virulence, is a first step towards this understanding. Reverse transcription quantitative real-time PCR (RT-qPCR) can be used to identify these candidate virulence genes, but relies on the use of reference genes for relative gene expression data normalisation. However, no report that addresses selecting appropriate fungal reference genes for use in the N. ditissima-apple pathosystem has been published to date. In this study, eight N. ditissima genes were selected as candidate RT-qPCR reference genes for gene expression analysis. A subset of the primers (six) designed to amplify regions from these genes were specific for N. ditissima, failing to amplify PCR products with template from other fungal pathogens present in the apple orchard. The efficiency of amplification of these six primer sets was satisfactory, ranging from 81.8 to 107.53%. Analysis of expression stability when a highly pathogenic N. ditissima isolate was cultured under 10 regimes, using the statistical algorithms geNorm, NormFinder and BestKeeper, indicated that actin and myo-inositol-1-phosphate synthase (mips), or their combination, could be utilised as the most suitable reference genes for normalisation of N. ditissima gene expression. As a test case, these reference genes were used to study expression of three candidate virulence genes during a time course of infection. All three, which shared traits with fungal effector genes, had up-regulated expression in planta compared to in vitro with expression peaking between five and six weeks post inoculation (wpi). Thus, these three genes may well be involved in N. ditissima pathogenicity and are priority candidates for further functional characterization.

Ten-Year Results of FAST: A Randomized Controlled Trial of 5-Fraction Whole-Breast Radiotherapy for Early Breast Cancer.

PURPOSE: Previous studies of hypofractionated adjuvant whole-breast radiotherapy for early breast cancer established a 15- or 16-fraction (fr) regimen as standard. The FAST Trial (CRUKE/04/015) evaluated normal tissue effects (NTE) and disease outcomes after 5-fr regimens. Ten-year results are presented. METHODS: Women ≥ 50 years of age with low-risk invasive breast carcinoma (pT1-2 pN0) were randomly assigned to 50 Gy/25 fr (5 weeks) or 30 or 28.5 Gy in 5 once-weekly fr of 6.0 or 5.7 Gy. The primary end point was change in photographic breast appearance at 2 and 5 years; secondary end points were physician assessments of NTE and local tumor control. Odds ratios (ORs) from longitudinal analyses compared regimens. RESULTS: A total of 915 women were recruited from 18 UK centers (2004-2007). Five-year photographs were available for 615/862 (71%) eligible patients. ORs for change in photographic breast appearance were 1.64 (95% CI, 1.08 to 2.49; P = .019) for 30 Gy and 1.10 (95% CI, 0.70 to 1.71; P = .686) for 28.5 Gy versus 50 Gy. α/ß estimate for photographic end point was 2.7 Gy (95% CI, 1.5 to 3.9 Gy), giving a 5-fr schedule of 28 Gy (95% CI, 26 to 30 Gy) estimated to be isoeffective with 50 Gy/25 fr. ORs for any moderate/marked physician-assessed breast NTE (shrinkage, induration, telangiectasia, edema) were 2.12 (95% CI, 1.55 to 2.89; P < .001) for 30 Gy and 1.22 (95% CI, 0.87 to 1.72; P = .248) for 28.5 Gy versus 50 Gy. With 9.9 years median follow-up, 11 ipsilateral breast cancer events (50 Gy: 3; 30 Gy: 4; 28.5 Gy: 4) and 96 deaths (50 Gy: 30; 30 Gy: 33; 28.5 Gy: 33) have occurred. CONCLUSION: At 10 years, there was no significant difference in NTE rates after 28.5 Gy/5 fr compared with 50 Gy/25 fr, but NTE were higher after 30 Gy/5 fr. Results confirm the published 3-year findings that a once-weekly 5-fr schedule of whole-breast radiotherapy can be identified that appears to be radiobiologically comparable for NTE to a conventionally fractionated regimen.

Genetic control of α-farnesene production in apple fruit and its role in fungal pathogenesis.

Terpenes are important compounds in plant trophic interactions. A meta-analysis of GC-MS data from a diverse range of apple (Malus × domestica) genotypes revealed that apple fruit produces a range of terpene volatiles, with the predominant terpene being the acyclic branched sesquiterpene (E,E)-α-farnesene. Four quantitative trait loci (QTLs) for α-farnesene production in ripe fruit were identified in a segregating 'Royal Gala' (RG) × 'Granny Smith' (GS) population with one major QTL on linkage group 10 co-locating with the MdAFS1 (α-farnesene synthase-1) gene. Three of the four QTLs were derived from the GS parent, which was consistent with GC-MS analysis of headspace and solvent-extracted terpenes showing that cold-treated GS apples produced higher levels of (E,E)-α-farnesene than RG. Transgenic RG fruit downregulated for MdAFS1 expression produced significantly lower levels of (E,E)-α-farnesene. To evaluate the role of (E,E)-α-farnesene in fungal pathogenesis, MdAFS1 RNA interference transgenic fruit and RG controls were inoculated with three important apple post-harvest pathogens [Colletotrichum acutatum, Penicillium expansum and Neofabraea alba (synonym Phlyctema vagabunda)]. From results obtained over four seasons, we demonstrate that reduced (E,E)-α-farnesene is associated with decreased disease initiation rates of all three pathogens. In each case, the infection rate was significantly reduced 7 days post-inoculation, although the size of successful lesions was comparable with infections on control fruit. These results indicate that (E,E)-α-farnesene production is likely to be an important factor involved in fungal pathogenesis in apple fruit.

Whole Genome Sequence Resource of the Asian Pear Scab Pathogen Venturia nashicola.

Venturia nashicola, the cause of scab disease of Asian pears, is a host-specific, biotrophic fungus. It is restricted to Asia and is regarded as a quarantine threat outside this region. European pear displays nonhost resistance (NHR) to V. nashicola and Asian pears are nonhosts of V. pyrina (the cause of European pear scab disease). The host specificity of these two fungi is likely governed by differences in their effector arsenals, with a subset hypothesized to activate NHR. The Pyrus-Venturia pathosystem provides an opportunity to dissect the underlying genetics of nonhost interactions in this potentially more durable form of resistance. The V. nashicola genome will enable comparisons to other Venturia spp. genomes to identify effectors that potentially activate NHR in the pear scab pathosystem.

Evidence for Sexual Reproduction: Identification, Frequency, and Spatial Distribution of Venturia effusa (Pecan Scab) Mating Type Idiomorphs.

Venturia effusa (syn. Fusicladium effusum), causal agent of pecan scab, is the most prevalent pathogen of pecan (Carya illinoinensis), causing severe yield losses in the southeastern United States. V. effusa is currently known only by its asexual (conidial) stage. However, the degree and distribution of genetic diversity observed within and among populations of V. effusa are typical of a sexually reproducing fungal pathogen, and comparable with other dothideomycetes with a known sexual stage, including the closely related apple scab pathogen, V. inaequalis. Using the mating type (MAT) idiomorphs from V. inaequalis, we identified a single MAT gene, MAT1-1-1, in a draft genome of V. effusa. The MAT1-1-1 locus is flanked by two conserved genes encoding a DNA lyase (APN2) and a hypothetical protein. The MAT locus spanning the flanking genes was amplified and sequenced from a subset of 14 isolates, of which 7 contained MAT1-1-1 and the remaining samples contained MAT1-2-1. A multiplex polymerase chain reaction screen was developed to amplify MAT1-1-1, MAT1-2-1, and a conserved reference gene encoding ß-tubulin, and used to screen 784 monoconidial isolates of V. effusa collected from 11 populations of pecan across the southeastern United States. A hierarchical sampling protocol representing region, orchard, and tree allowed for analysis of MAT structure at different spatial scales. Analysis of this collection revealed the frequency of the MAT idiomorphs is in a 1:1 equilibrium of MAT1-1:MAT1-2. The apparent equilibrium of the MAT idiomorphs provides impetus for a renewed effort to search for the sexual stage of V. effusa. [Formula: see text] Copyright © 2018 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Comparative analysis of the predicted secretomes of Rosaceae scab pathogens Venturia inaequalis and V. pirina reveals expanded effector families and putative determinants of host range.

BACKGROUND: Fungal plant pathogens belonging to the genus Venturia cause damaging scab diseases of members of the Rosaceae. In terms of economic impact, the most important of these are V. inaequalis, which infects apple, and V. pirina, which is a pathogen of European pear. Given that Venturia fungi colonise the sub-cuticular space without penetrating plant cells, it is assumed that effectors that contribute to virulence and determination of host range will be secreted into this plant-pathogen interface. Thus the predicted secretomes of a range of isolates of Venturia with distinct host-ranges were interrogated to reveal putative proteins involved in virulence and pathogenicity. RESULTS: Genomes of Venturia pirina (one European pear scab isolate) and Venturia inaequalis (three apple scab, and one loquat scab, isolates) were sequenced and the predicted secretomes of each isolate identified. RNA-Seq was conducted on the apple-specific V. inaequalis isolate Vi1 (in vitro and infected apple leaves) to highlight virulence and pathogenicity components of the secretome. Genes encoding over 600 small secreted proteins (candidate effectors) were identified, most of which are novel to Venturia, with expansion of putative effector families a feature of the genus. Numerous genes with similarity to Leptosphaeria maculans AvrLm6 and the Verticillium spp. Ave1 were identified. Candidates for avirulence effectors with cognate resistance genes involved in race-cultivar specificity were identified, as were putative proteins involved in host-species determination. Candidate effectors were found, on average, to be in regions of relatively low gene-density and in closer proximity to repeats (e.g. transposable elements), compared with core eukaryotic genes. CONCLUSIONS: Comparative secretomics has revealed candidate effectors from Venturia fungal plant pathogens that attack pome fruit. Effectors that are putative determinants of host range were identified; both those that may be involved in race-cultivar and host-species specificity. Since many of the effector candidates are in close proximity to repetitive sequences this may point to a possible mechanism for the effector gene family expansion observed and a route to diversification via transposition and repeat-induced point mutation.

Variation in Host and Pathogen in the Neonectria/Malus Interaction; toward an Understanding of the Genetic Basis of Resistance to European Canker.

Apple canker caused by the phytopathogenic fungus Neonectria ditissima is an economically important disease, which has spread in recent years to almost all pome-producing regions of the world. N. ditissima is able to cross-infect a wide range of apple varieties and causes branch and trunk lesions, known as cankers. Most modern apple varieties are susceptible and in extreme cases suffer from high mortality (up to 50%) in the early phase of orchard establishment. There is no known race structure of the pathogen and the global level of genetic diversity of the pathogen population is unknown. Resistance breeding is underway in many global breeding programmes, but nevertheless, a total resistance to canker has not yet been demonstrated. Here we present preliminary data from a survey of the phylogenetic relationships between global isolates of N. ditissima which reveals only slight evidence for population structure. In addition we report the results of four rapid screening tests to assess the response to N. ditissima in different apple scion and rootstock varieties, which reveals abundant variation in resistance responses in both cultivar and rootstock material. Further seedling tests show that the segregation patterns of resistance and susceptibility vary widely between crosses. We discuss inconsistencies in test performance with field observations and discuss future research opportunities in this area.

A Large Family of AvrLm6-like Genes in the Apple and Pear Scab Pathogens, Venturia inaequalis and Venturia pirina.

Venturia inaequalis and V. pirina are Dothideomycete fungi that cause apple scab and pear scab disease, respectively. Whole genome sequencing of V. inaequalis and V. pirina isolates has revealed predicted proteins with sequence similarity to AvrLm6, a Leptosphaeria maculans effector that triggers a resistance response in Brassica napus and B. juncea carrying the resistance gene, Rlm6. AvrLm6-like genes are present as large families (>15 members) in all sequenced strains of V. inaequalis and V. pirina, while in L. maculans, only AvrLm6 and a single paralog have been identified. The Venturia AvrLm6-like genes are located in gene-poor regions of the genomes, and mostly in close proximity to transposable elements, which may explain the expansion of these gene families. An AvrLm6-like gene from V. inaequalis with the highest sequence identity to AvrLm6 was unable to trigger a resistance response in Rlm6-carrying B. juncea. RNA-seq and qRT-PCR gene expression analyses, of in planta- and in vitro-grown V. inaequalis, has revealed that many of the AvrLm6-like genes are expressed during infection. An AvrLm6 homolog from V. inaequalis that is up-regulated during infection was shown (using an eYFP-fusion protein construct) to be localized to the sub-cuticular stroma during biotrophic infection of apple hypocotyls.

Repeat-containing protein effectors of plant-associated organisms.

Many plant-associated organisms, including microbes, nematodes, and insects, deliver effector proteins into the apoplast, vascular tissue, or cell cytoplasm of their prospective hosts. These effectors function to promote colonization, typically by altering host physiology or by modulating host immune responses. The same effectors however, can also trigger host immunity in the presence of cognate host immune receptor proteins, and thus prevent colonization. To circumvent effector-triggered immunity, or to further enhance host colonization, plant-associated organisms often rely on adaptive effector evolution. In recent years, it has become increasingly apparent that several effectors of plant-associated organisms are repeat-containing proteins (RCPs) that carry tandem or non-tandem arrays of an amino acid sequence or structural motif. In this review, we highlight the diverse roles that these repeat domains play in RCP effector function. We also draw attention to the potential role of these repeat domains in adaptive evolution with regards to RCP effector function and the evasion of effector-triggered immunity. The aim of this review is to increase the profile of RCP effectors from plant-associated organisms.

Draft Genome Sequences of Two Isolates of the Plant-Pathogenic Fungus Neonectria ditissima That Differ in Virulence.

Neonectria ditissima is the causal agent of apple canker. Here, we present the draft genome sequences of two isolates of N. ditissima that differ in virulence. Comparative genomics will enable pathogenicity determinants to be identified in this plant-pathogenic fungus.

Proteogenomic analysis of the Venturia pirina (Pear Scab Fungus) secretome reveals potential effectors.

A proteogenomic analysis is presented for Venturia pirina, a fungus that causes scab disease on European pear (Pyrus communis). V. pirina is host-specific, and the infection is thought to be mediated by secreted effector proteins. Currently, only 36 V. pirina proteins are catalogued in GenBank, and the genome sequence is not publicly available. To identify putative effectors, V. pirina was grown in vitro on and in cellophane sheets mimicking its growth in infected leaves. Secreted extracts were analyzed by tandem mass spectrometry, and the data (ProteomeXchange identifier PXD000710) was queried against a protein database generated by combining in silico predicted transcripts with six frame translations of a whole genome sequence of V. pirina (GenBank Accession JEMP00000000 ). We identified 1088 distinct V. pirina protein groups (FDR 1%) including 1085 detected for the first time. Thirty novel (not in silico predicted) proteins were found, of which 14 were identified as potential effectors based on characteristic features of fungal effector protein sequences. We also used evidence from semitryptic peptides at the protein N-terminus to corroborate in silico signal peptide predictions for 22 proteins, including several potential effectors. The analysis highlights the utility of proteogenomics in the study of secreted effectors.