Biology subjects

Mesarich, C. H.

Publications and source records attributed to Mesarich, C. H..

At least 19 recordsLinked to original sources

bioRxiv2025

From Nicotiana to apple: effector screening reveals new avenues for durable scab resistance

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 NbSOBIR1 or NbBAK1, 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 VmE02 effector protein from the apple pathogen Valsa mali, did so in an NbSOBIR1- and NbBAK1-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.

microbiology↗

bioRxiv2025

Ancient MAX Effector Variants of a Fungal Pathogen Evade Apoplastic Immunity in Apple

Venturia inaequalis is an Ascomycota fungus responsible for apple scab, the main disease in apple orchards. During infection, the pathogen colonizes the subcuticular space and secretes effectors to promote virulence. To mitigate disease impact, resistance genes such as Rvi6, which encodes a membrane-localized receptor-like protein, have been introduced into apple cultivars. However, over the past three decades, Rvi6-mediated resistance has been circumvented in orchards, with the emergence of virulent V. inaequalis strains. Through comparative genomics analyses and functional validation by complementation, we identified AvrRvi6 as the fungal determinant that activates Rvi6-mediated immunity. Screening 122 V. inaequalis strains worldwide, we identified 20 distinct AvrRvi6 alleles, of which only five are not recognized by Rvi6. Evolution analysis demonstrated that the emergence of these virulent alleles predates the domestication of apple, thus revealing that wild Malus species constitute a reservoir of virulence. Using AlphaFold structural modeling, we showed that AvrRvi6 belongs to an expanded family that adopts a MAX effector fold, originally described for cytoplasmic effectors of the blast fungus Pyricularia oryzae. Transient expression in Nicotiana benthamiana demonstrated that AvrRvi6 triggers allele-specific apoplastic immunity and validated three different molecular mechanisms (mutation, partial deletion and transposon insertion in the promoter) that V. inaequalis employs to circumvent Rvi6 recognition. This study delivers the first characterization of a V. inaequalis avirulence factor, uncovers an unexpected apoplastic role for a MAX effector, and demonstrates how ancestral virulence diversity maintained in wild populations compromises resistance durability in apple.

microbiology↗

bioRxiv2025

PaRXLR40, a broad cell death suppressor of the kauri dieback pathogen Phytophthora agathidicida, targets a plant ARM/BTB domain-containing protein

O_LIPhytophthora agathidicida, the causal agent of kauri dieback, secretes RXLR effector proteins to promote host colonisation. One of these, PaRXLR40, was previously shown to suppress immune responses in Nicotiana benthamiana, but its mechanism of action and contribution to virulence remained unclear. C_LIO_LITo investigate PaRXLR40 function, we used comparative approaches in N. benthamiana and Agathis australis (kauri), including RNA interference (RNAi), transient expression assays, confocal microscopy, yeast two-hybrid screens, and infection assays. We also examined host protein interactors and tested mutant variants to evaluate functional domains. C_LIO_LISilencing PaRXLR40 reduced P. agathidicida colonization in N. benthamiana and A. australis. PaRXLR40 interacted with a host BTB/ARM domain protein (ARIA), previously implicated in abscisic acid (ABA) signalling. ARIA suppressed immunity and promoted infection, while interacting with NbSOG1, a DNA damage-associated transcription factor that enhanced resistance when overexpressed. External application of ABA enhanced P. agathidicida infection in both hosts, supporting the hypothesis that PaRXLR40 may hijack host ABA signalling through ARIA to promote susceptibility. C_LIO_LIOur findings show that PaRXLR40 targets ARIA to manipulate host immunity and promote virulence. The interaction between ARIA and SOG1 suggests PaRXLR40 may interfere with host transcriptional reprogramming. PaRXLR40 represents a potential target for future RNAi-based strategies to reduce kauri dieback. C_LI

molecular biology↗

bioRxiv2025

Mutation or deletion of the Avr6 avirulence effector gene of Fulvia fulva leads to breakdown of the Cf-6 leaf mold resistance locus in tomato

Genetic resistance mediated by Cf cell-surface receptors is a cornerstone of tomato breeding against leaf mold disease caused by the fungal pathogen Fulvia fulva. After widespread deployment of the Cf-9 resistance locus, breeders increasingly relied on Cf-6, yet Cf-6-mediated resistance has already been overcome in multiple regions. The molecular basis of this breakdown, and the identity of the matching avirulence (Avr) effector, have remained unresolved. Here, we use comparative genomics and functional genetics to identify the F. fulva effector Avr6 as the previously described apoplastic protein Ecp5. Sequencing of Cf-6-breaking strains revealed either deletion of the Ecp5 locus or non-synonymous mutations in its coding sequence. Using transient expression, targeted gene knockout, and complementation assays, we show that Ecp5 is both necessary and sufficient for Cf-6-mediated recognition and defence activation. We then use Avr6-triggered cell death as a phenotypic marker in a bulked segregant analysis combined with Comparative Subsequence Sets analysis (CoSSa) to map Cf-6 to a [~]2 Mb interval on the short arm of chromosome 12 that overlaps the previously described Cf-Ecp5.12 locus. Sequencing of Avr6 in a broad strain collection reveals a predominantly conserved allele under apparent purifying selection, with recent independent loss-of-function variants in Europe and South America that confer virulence on Cf-6 plants. Together, these findings establish the Avr6-Cf-6 gene-for-gene pair, explain the erosion of Cf-6-mediated resistance, and illustrate how effector loss or mutation provides a rapid route for F. fulva adaptation, with important implications for designing more durable resistance strategies against tomato leaf mold.

microbiology↗

bioRxiv2025

Molecular investigation of Rlm3 from rapeseed as a broad-spectrum resistance gene against fungal pathogens producing structurally conserved effectors

Recognition of a pathogen avirulence (AVR) effector protein by its cognate plant resistance (R) protein triggers immune responses that render the plant resistant, representing an efficient disease control strategy. While (AVR) effectors have long been considered species- or genotype-specific, based on a lack of homology in sequence databases, a growing number of studies have now shown that these proteins belong to a limited set of structural families. This is an important finding because it paves the way for the identification or engineering of broad- spectrum R proteins capable of recognizing several members of the same structural family. In the Leptosphaeria maculans / rapeseed (Brassica napus) pathosystem, 13 AVR genes have been cloned, of which four encode effectors belonging to the LARS (Leptosphaeria AviRulence and Supressing) structural family that has also been found in thirteen other phytopathogenic fungi. Homologues of the L. maculans AvrLm3 AVR protein, a LARS family member, have been identified in other fungal species, including an AVR protein from Fulvia fulva called Ecp11-1. We have previously shown that Ecp11-1 is recognized by rapeseed varieties carrying the Rlm3 R gene, and that this recognition is masked by the presence of another LARS AVR gene, AvrLm4-7. In this study, we expanded our characterization of the Rlm3 resistance spectrum to putative effectors from Fusarium oxysporum and Zymoseptoria ardabiliae and showed that one effector from F. oxysporum f. sp. narcissi behaves like Ecp11-1, being recognized by Rlm3, and this recognition also being masked by the presence of AvrLm4-7. We finally investigated which protein regions and amino acids were necessary for the recognition of AvrLm3 and Ecp11-1 by Rlm3. This analysis is a first step towards the identification or engineering of broad- spectrum R proteins that confer protection against multiple phytopathogens through the recognition of structural effector families.

plant biology↗

bioRxiv2023

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

O_LILeaf 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. C_LIO_LIComparative 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. C_LIO_LIA 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. C_LIO_LIThis 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. C_LI

microbiology↗

bioRxiv2023

Genomic, effector protein and culture-based analysis of Cyclaneusma minus in New Zealand provides evidence for multiple morphotypes

Cyclaneusma needle cast, caused by Cyclaneusma minus, affects Pinus species around the world. 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 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 and some family members being found exclusively in some morphotypes. We also showed that proteins belonging to the Ecp32 family trigger cell death responses in non-host Nicotiana species, and, as previously demonstrated for other plant pathogens, the C. minus proteins belonging to the Ecp32 family adopt a {beta}-trefoil fold. Understanding the geographical range and variations in virulence and pathogenicity of these morphotypes will provide a better understanding of pine needle diseases as well as enable the development of more durable methods to control this disease.

molecular biology↗

bioRxiv2022

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 defences 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 {beta}-1,6-glucan, are down-regulated 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 defences and recognition by the host immune system. ImportancePlant-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 down-regulates genes associated with the biosynthesis of two immunogenic cell wall carbohydrates, chitin and {beta}-1,6-glucan, and coats its infection structures with a less-immunogenic carbohydrate, chitosan. These changes are anticipated to enable subcuticular 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.

microbiology↗

bioRxiv2022

Characterization of two conserved cell death elicitor families from the Dothideomycete fungal pathogens Dothistroma septosporum and Fulvia fulva (syn. Cladosporium fulvum)

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 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 {beta}-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 {beta}-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.

molecular biology↗

bioRxiv2022

The Venturia inaequalis effector repertoire is expressed in waves, and is dominated by expanded families with predicted structural similarity to avirulence proteins from other fungi

BackgroundScab, 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. ResultsWe 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. ConclusionsOur 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.

microbiology↗

bioRxiv2021

Effector loss drives adaptation of Pseudomonas syringae pv. actinidiae to Actinidia arguta

A pandemic isolate of Pseudomonas syringae pv. actinidiae biovar 3 (Psa3) has devastated kiwifruit orchards growing cultivars of Actinidia chinensis. In contrast, A. arguta (kiwiberry) is resistant to Psa3. This resistance is mediated via effector-triggered immunity, as demonstrated by induction of the hypersensitive response in infected A. arguta leaves, observed by microscopy and quantified by ion-leakage assays. Isolates of Psa3 that cause disease in A. arguta have been isolated and analyzed, revealing a 49 kb deletion in the exchangeable effector locus (EEL). This natural EEL-mutant isolate and strains with synthetic knockouts of the EEL were more virulent in A. arguta plantlets than wild-type Psa3. Screening of a complete library of Psa3 effector knockout strains identified increased growth in planta for knockouts of four effectors - AvrRpm1a, HopF1c, HopZ5a, and the EEL effector HopAW1a - suggesting a resistance response in A. arguta. Hypersensitive response (HR) assays indicate that three of these effectors trigger a host species-specific HR. A Psa3 strain with all four effectors knocked out escaped host recognition, but a cumulative increase in bacterial pathogenicity and virulence was not observed. These avirulence effectors can be used in turn to identify the first cognate resistance genes in Actinidia for breeding durable resistance into future kiwifruit cultivars.

plant biology↗

bioRxiv2021

Apoplastic effector candidates of a foliar forest pathogen trigger cell death in host and non-host plants

Forests are under threat from pests, pathogens, and changing climate. One of the major forest pathogens worldwide is Dothistroma septosporum, which causes dothistroma needle blight (DNB) of pines. D. septosporum is a hemibiotrophic fungus related to well-studied Dothideomycete pathogens, such as Cladosporium fulvum. These pathogens use small secreted proteins, termed effectors, to facilitate the infection of their hosts. The same effectors, however, can be recognised by plants carrying corresponding immune receptors, resulting in resistance responses. Hence, effectors are increasingly being exploited to identify and select disease resistance in crop species. In gymnosperms, however, such research is scarce. We predicted and investigated apoplastic D. septosporum candidate effectors (DsCEs) using bioinformatics and plant-based experiments. We discovered secreted proteins that trigger cell death in the angiosperm Nicotiana spp., suggesting their recognition by immune receptors in non-host plants. In a first for foliar forest pathogens, we also developed a novel protein infiltration method to show that tissue-cultured pine shoots can respond with a cell death response to one of our DsCEs, as well as to a reference cell death-inducing protein. These results contribute to our understanding of forest pathogens and may ultimately provide clues to disease immunity in both commercial and natural forests.

plant biology↗

bioRxiv2021

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

BackgroundScab, or black spot, caused by the filamentous 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. Recently, CRISPR-Cas9 has emerged as an efficient tool for gene editing in filamentous fungi. With this in mind, we set out to establish CRISPR-Cas9 as a gene editing tool in V. inaequalis. ResultsWe showed that CRISPR-Cas9 can be used for gene inactivation in the apple scab fungus. As a proof of concept, we targeted the melanin biosynthesis pathway gene trihydroxynaphthalene reductase (THN), which has previously been shown to result in a light-brown colony phenotype when transcriptionally silenced using RNA interference. Using one of two CRISPR-Cas9 single guide RNAs (sgRNAs) 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 these 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. ConclusionsIn establishing CRISPR-Cas9 as a tool for gene editing in V. inaequalis, we have provided a strong starting point for studies aiming to decipher the function of genes associated with the growth, reproduction, virulence and pathogenicity of 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.

microbiology↗

bioRxiv2020

A new family of structurally conserved fungal effectors displays epistatic interactions with plant resistance proteins

Recognition of a pathogen avirulence (AVR) effector protein by a cognate plant resistance (R) protein triggers a set of immune responses that render the plant resistant. Pathogens can escape this so-called Effector-Triggered Immunity (ETI) by different mechanisms including the deletion or loss-of-function mutation of the AVR gene, the incorporation of point mutations that allow recognition to be evaded while maintaining virulence function, and the acquisition of new effectors that suppress AVR recognition. The Dothideomycete Leptosphaeria maculans, causal agent of oilseed rape stem canker, is one of the few fungal pathogens where suppression of ETI by an AVR effector has been demonstrated. Indeed, AvrLm4-7 suppresses Rlm3- and Rlm9- mediated resistance triggered by AvrLm3 and AvrLm5-9, respectively. The presence of AvrLm4-7 does not impede AvrLm3 and AvrLm5-9 expression, and the three AVR proteins do not appear to physically interact. To decipher the epistatic interaction between these L. maculans AVR effectors, we determined the crystal structure of AvrLm5-9 and obtained a 3D model of AvrLm3, based on the crystal structure of Ecp11-1, a homologous AVR effector candidate from Fulvia fulva. Despite a lack of sequence similarity, AvrLm5-9 and AvrLm3 are structural analogues of AvrLm4-7 (structure previously characterized). Structure-informed sequence database searches identified a larger number of putative structural analogues among L. maculans effector candidates, including the AVR effector AvrLmS-Lep2, all produced during the early stages of oilseed rape infection, as well as among effector candidates from other phytopathogenic fungi. These structural analogues are named LARS (for Leptosphaeria AviRulence and Suppressing) effectors. Remarkably, transformants of L. maculans expressing one of these structural analogues, Ecp11-1, triggered oilseed rape immunity in several genotypes carrying Rlm3. Furthermore, this resistance could be suppressed by AvrLm4-7. These results suggest that Ecp11-1 shares a common activity with AvrLm3 within the host plant which is detected by Rlm3, or that the Ecp11-1 structure is sufficiently close to that of AvrLm3 to be recognized by Rlm3. Author summaryAn efficient strategy to control fungal diseases in the field is genetic control using resistant crop cultivars. Crop resistance mainly relies on gene-for-gene relationships between plant resistance (R) genes and pathogen avirulence (AVR) genes, as defined by Flor in the 1940s. However, such gene-for-gene relationships can increase in complexity over the course of plant-pathogen co-evolution. Resistance against the plant-pathogenic fungus Leptosphaeria maculans by Brassica napus and other Brassica species relies on the recognition of effector (AVR) proteins by R proteins; however, L. maculans produces an effector that suppresses a subset of these specific resistances. Using a protein structure approach, we revealed structural analogy between several of the resistance-triggering effectors, the resistance-suppressing effector, and effectors from other plant-pathogenic species in the Dothideomycetes and Sordariomycetes classes, defining a new family of effectors called LARS. Notably, cross-species expression of one LARS effector from Fulvia fulva, a pathogen of tomato, in L. maculans resulted in recognition by several resistant cultivars of oilseed rape. These results highlight the need to integrate knowledge on effector structures to improve resistance management and to develop broad-spectrum resistances for multi-pathogen control of diseases.

plant biology↗

bioRxiv2020

A homologue of the mammalian tumour suppressor protein PTEN is a functional lipid phosphatase and required for chemotaxis in filamentous fungi

Phosphoinositides (PI) are essential components of eukaryotic membranes and function in a large number of signalling processes. While lipid second messengers are well studied in mammals and yeast, their role in filamentous fungi is poorly understood. We used fluorescent PI-binding molecular probes to localise the phosphorylated phosphatidylinositol species PI[3]P, PI[3,5]P2, PI[4]P and PI[4,5]P2 in hyphae of the endophyte Epichloe festucae in axenic culture and during interaction with its grass host Lolium perenne. We also analysed the roles of the phosphatidylinositol-4-phosphate 5-kinase MssD and the predicted phosphatidylinositol-3,4,5-triphosphate 3-phosphatase TepA, a homologue of the mammalian tumour suppressor protein PTEN. Deletion of tepA in E. festucae and in the root-infecting tomato pathogen Fusarium oxysporum had no impact on growth in culture or the host interaction phenotype. However, this mutation did uncover the presence of PI[3,4,5]P3 in septa of E. festucae and showed that TepA is required for chemotropism in F. oxysporum. The identification of PI[3,4,5]P3 in septa of{Delta} tepA strains suggests that filamentous fungi are able to generate PI[3,4,5]P3 using an alternative biosynthetic pathway and that fungal PTEN homologues are functional lipid phosphatases. The F. oxysporum chemotropism defect demonstrates a conserved role of PTEN homologues in chemotaxis across protists, fungi and mammals.

cell biology↗

bioRxiv2020

Variation at the common polysaccharide antigen locus drives lipopolysaccharide diversity within the P. syringae species complex

The common polysaccharide antigen (CPA) from the lipopolysaccharide (LPS) component of cell walls from the species complex Pseudomonas syringae is highly variable both in structure and immunological specificity, but the genetic basis for this is not well understood. We have characterised the CPA locus from P. syringae pv. actinidiae (Psa). This locus has a modular structure with genes for both L- and D- rhamnose (Rha) biosynthesis and that of an unknown sugar. It also contains an operon coding for ABC transporter subunits, a bifunctional glycosyltransferase and an O-methyltransferase. This operon is predicted to have a role in transport, elongation and termination of the Rha backbone of the CPA oligosaccharide and is referred to as the TET operon. This is the first report of the identification of this operon in P. syringae. Two alleles of the TET operon were present amongst the different biovars of Psa and lineages of the closely related pathovar P. syringae pv. actinidifoliorum. This allelic variation was reflected in the electrophoretic properties of purified LPS from the different isolates. Gene knockout of the TET operon allele from biovar 1 and replacement with that from biovar 3, demonstrated the link between the genetic locus and the electrophoretic and immunogenic properties of the LPS molecules in Psa. Sequence analysis of the TET operon from a wide range of P. syringae and P. viridiflava isolates displayed a phylogenetic history which is incongruent with core gene phylogeny, but correlates with previously reported tailocin sensitivity, suggesting a functional relationship between LPS structure and tailocin susceptibility.

microbiology↗

bioRxiv2019

Reprogramming of the apoplast metabolome of Lolium perenne upon infection with the mutualistic symbiont Epichloë festucae

Epichloe festucae is an endophytic fungus that forms a mutualistic symbiotic association with Lolium perenne. Here we analysed how the metabolome of the ryegrass apoplast changed upon infection of this host with sexual and asexual isolates of E. festucae. A metabolite fingerprinting approach was used to analyse the metabolite composition of apoplastic wash fluid from non-infected and infected L. perenne. Metabolites enriched or depleted in one or both of these treatments were identified using a set of interactive tools. A genetic approach in combination with tandem mass spectrometry was used to identify a novel product of a secondary metabolite gene cluster. Metabolites likely to be present in the apoplast were identified using the MarVis Pathway in combination with the BioCyc and KEGG databases, and an in-house Epichloe metabolite database. We were able to identify the known endophyte-specific metabolites, peramine and epichloecyclins, as well as a large number of unknown markers. To determine whether these methods can be applied to the identification of novel Epichloe-derived metabolites, we deleted a gene encoding a NRPS (lgsA) that is highly expressed in planta. Comparative mass spectrometric analysis of apoplastic wash fluid from wild-type- versus mutant- infected plants identified a novel Leu/Ile glycoside metabolite present in the former.

microbiology↗

bioRxiv2019

Phosphatidic acid produced by phospholipase D is required for hyphal cell-cell fusion and fungal-plant symbiosis

Although lipid signaling has been shown to serve crucial roles in mammals and plants, little is known about this process in filamentous fungi. Here we analyse the contribution of phospholipase D (PLD) and its product phosphatidic acid (PA) in hyphal morphogenesis and growth of Epichloe festucae and Neurospora crassa, and in the establishment of a symbiotic interaction between E. festucae and Lolium perenne. Growth of E. festucae and N. crassa PLD deletion strains in axenic culture, and for E. festucae in association with L. perenne, were analysed by light-, confocal- and electron microscopy. Changes in PA distribution were analysed in E. festucae using a PA biosensor and the impact of these changes on endocytic recycling and superoxide production investigated. We found that E. festucae PldB and the N. crassa ortholog, PLA-7, are required for polarized growth, cell fusion and ascospore development, whereas PldA/PLA-8 are dispensable for these functions. Exogenous addition of PA rescues the cell-fusion phenotype in E. festucae. PldB is also crucial for E. festucae to establish a symbiotic association with L. perenne. This study identifies a new component of the cell-cell communication and cell fusion signaling network that controls hyphal morphogenesis and growth in filamentous fungi.

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