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Background: Compatibility between plant parasites and their hosts is genetically determined {Citation}both interacting organisms. For example, plants may carry resistance (R) genes or deploy chemical defences. Aphid saliva contains many proteins that are secreted into host tissues. Subsets of these proteins are predicted to act as effectors, either subverting or triggering host immunity. However, associating particular effectors with virulence or avirulence outcomes presents challenges due to the combinatorial complexity. Here we use defined aphid and host genetics to test for co-segregation of expressed aphid transcripts and proteins with virulent or avirulent phenotypes., Results: We compared virulent and avirulent pea aphid parental genotypes, and their bulk segregant F1 progeny on Medicago truncatula genotypes carrying or lacking the RAP1 (Resistance to Acyrthosiphon pisum 1) resistance quantitative trait locus. Differential gene expression analysis of whole body and head samples, in combination with proteomics of saliva and salivary glands, enabled us to pinpoint proteins associated with virulence or avirulence phenotypes. There was relatively little impact of host genotype, whereas large numbers of transcripts and proteins were differentially expressed between parental aphids, likely a reflection of their classification as divergent biotypes within the pea aphid species complex. Many fewer transcripts intersected with the equivalent differential expression patterns in the bulked F1 progeny, providing an effective filter for removing genomic background effects. Overall, there were more upregulated genes detected in the F1 avirulent dataset compared with the virulent one. Some genes were differentially expressed both in the transcriptome and in the proteome datasets, with aminopeptidase N proteins being the most frequent differentially expressed family. In addition, a substantial proportion (27%) of salivary proteins lack annotations, suggesting that many novel functions remain to be discovered., Conclusions: Especially when combined with tightly controlled genetics of both insect and host plant, multi-omics approaches are powerful tools for revealing and filtering candidate lists down to plausible genes for further functional analysis as putative aphid effectors., Competing Interests: Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Barley net form net blotch (NFNB) is a destructive foliar disease caused by Pyrenophora teres f. teres. Barley line CIho5791, which harbors the broadly effective chromosome 6H resistance gene Rpt5 , displays dominant resistance to P. teres f. teres . To genetically characterize P. teres f. teres avirulence/virulence on the barley line CIho5791, we generated a P. teres f. teres mapping population using a cross between the Moroccan CIho5791-virulent isolate MorSM40-3 and the avirulent reference isolate 0-1. Full genome sequences were generated for 103 progenies. Saturated chromosome-level genetic maps were generated, and quantitative trait locus (QTL) mapping identified two major QTL associated with P. teres f. teres avirulence/virulence on CIho5791. The most significant QTL mapped to chromosome (Ch) 1, where the virulent allele was contributed by MorSM40-3. A second QTL mapped to Ch8; however, this virulent allele was contributed by the avirulent parent 0-1. The Ch1 and Ch8 loci accounted for 27 and 15% of the disease variation, respectively, and the avirulent allele at the Ch1 locus was epistatic over the virulent allele at the Ch8 locus. As a validation, we used a natural P. teres f. teres population in a genome-wide association study that identified the same Ch1 and Ch8 loci. We then generated a new reference quality genome assembly of parental isolate MorSM40-3 with annotation supported by deep transcriptome sequencing of infection time points. The annotation identified candidate genes predicted to encode small, secreted proteins, one or more of which are likely responsible for overcoming the CIho5791 resistance. [Formula: see text] The author(s) have dedicated the work to the public domain under the Creative Commons CC0 "No Rights Reserved" license by waiving all of his or her rights to the work worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law, 2024., Competing Interests: The author(s) declare no conflict of interest.

Grapevine downy mildew, caused by the oomycete Plasmopara viticola (P. viticola, Berk. & M. A. Curtis; Berl. & De Toni), is a global threat to Eurasian wine grapes Vitis vinifera. Although resistant grapevine varieties are becoming more accessible, P. viticola populations are rapidly evolving to overcome these resistances. We aimed to uncover avirulence genes related to Rpv3.1-mediated grapevine resistance. We sequenced the genomes and characterized the development of 136 P. viticola strains on resistant and sensitive grapevine cultivars. A genome-wide association study was conducted to identify genomic variations associated with resistant-breaking phenotypes. We identified a genomic region associated with the breakdown of Rpv3.1 grapevine resistance (avrRpv3.1 locus). A diploid-aware reassembly of the P. viticola INRA-Pv221 genome revealed structural variations in this locus, including a 30 kbp deletion. Virulent P. viticola strains displayed multiple deletions on both haplotypes at the avrRpv3.1 locus. These deletions involve two paralog genes coding for proteins with 800-900 amino acids and signal peptides. These proteins exhibited a structure featuring LWY-fold structural modules, common among oomycete effectors. When transiently expressed, these proteins induced cell death in grapevines carrying Rpv3.1 resistance, confirming their avirulence nature. This discovery sheds light on the genetic mechanisms enabling P. viticola to adapt to grapevine resistance, laying a foundation for developing strategies to manage this destructive crop pathogen., (© 2024 The Authors. New Phytologist © 2024 New Phytologist Foundation.)

Verticillium wilt, caused by Verticillium dahliae , is one of the most devastating soilborne diseases of lettuce ( Lactuca sativa L.). There are three races of V. dahliae , and each race has been characterized by markers representing race-specific effectors. Race 1 is differentiated by the presence of the functional secretory Ave1 effector. Similarly, races 2 and 3 are differentiated by effectors VdR2e and VdR3e , respectively. Although the presence of race 1 in coastal California was well established, the presence of effector-based races 2 and 3 was uncertain. This study therefore focused on characterizing 727 isolates collected from 142 ranches of symptomatic lettuce and other crops from coastal California. Based on this evaluation, 523 isolates were designated as race 1, 20 isolates as race 2, 23 isolates as race 3, and 17 as race undefined. Isolates representing other Verticillium species totaled 110, and 34 were non- Verticillium fungal species. Because the use of resistant cultivars is a key strategy to manage this disease, we evaluated 48 lettuce germplasm lines and 1 endive ( Cichorium endivia L.) line, comprising commercial cultivars and breeding lines, including the race 1-resistant heirloom cultivar La Brillante and the susceptible cultivar Salinas as controls. Resistance against races 1, 2, and 3 along with VdLs17, a virulent isolate of V. dahliae from lettuce that is currently not assigned to a race, was evaluated in replicated greenhouse experiments. Two crisphead lettuce lines, HL28 and HL29, exhibited resistance against race 1 and a partial resistance against race 2, whereas all other lines were highly susceptible to races 1 and 2 and VdLs17. The majority of lines exhibited higher resistance to race 3 relative to the other two races. This study documents the current distribution of the different races in coastal California. In addition, the sources of resistance currently being developed should be effective or partially effective against these races for targeted deployment as soon as they are available., Competing Interests: The author(s) declare no conflict of interest.

Uncovering the function of phytopathogen effectors is crucial for understanding mechanisms of pathogen pathogenicity and for improving our ability to protect plants from diseases. An increasing number of effectors have been predicted in various plant pathogens. Functional characterization of these effectors has become a major focus in the study of plant-pathogen interactions. In this study, we designed a novel screening system that combines the TMV (tobacco mosaic virus)-GFP vector and Agrobacterium-mediated transient expression in the model plant Nicotiana benthamiana. This system enables the rapid identification of effectors that interfere with plant immunity. The biological function of these effectors can be easily evaluated by observing the GFP fluorescence signal using a UV lamp within just a few days. To evaluate the TMV-GFP system, we initially tested it with well-described virulence and avirulence type III effectors from the bacterial pathogen Ralstonia solanacearum. After proving the accuracy and efficiency of the TMV-GFP system, we successfully screened a novel virulence effector, RipS1, using this approach. Furthermore, using the TMV-GFP system, we reproduced consistent results with previously known cytoplasmic effectors from a diverse array of pathogens. Additionally, we demonstrated the effectiveness of the TMV-GFP system in identifying apoplastic effectors. The easy operation, time-saving nature, broad effectiveness, and low technical requirements of the TMV-GFP system make it a promising approach for high-throughput screening of effectors with immune interference activity from various pathogens., (© 2024 Society for Experimental Biology and John Wiley & Sons Ltd.)

The powdery mildew fungi (Erysiphaceae) are globally distributed plant pathogens with a range of more than 10,000 plant hosts. In this review, we discuss the long- and short-term evolution of these obligate biotrophic fungi and outline their diversity with respect to morphology, lifestyle, and host range. We highlight their remarkable ability to rapidly overcome plant immunity, evolve fungicide resistance, and broaden their host range, for example, through adaptation and hybridization. Recent advances in genomics and proteomics, particularly in cereal powdery mildews (genus Blumeria), provided first insights into mechanisms of genomic adaptation in these fungi. Transposable elements play key roles in shaping their genomes, where even close relatives exhibit diversified patterns of recent and ongoing transposon activity. These transposons are ubiquitously distributed in the powdery mildew genomes, resulting in a highly adaptive genome architecture lacking obvious regions of conserved gene space. Transposons can also be neofunctionalized to encode novel virulence factors, particularly candidate secreted effector proteins, which may undermine the plant immune system. In cereals like barley and wheat, some of these effectors are recognized by plant immune receptors encoded by resistance genes with numerous allelic variants. These effectors determine incompatibility ("avirulence") and evolve rapidly through sequence diversification and copy number variation. Altogether, powdery mildew fungi possess plastic genomes that enable their fast evolutionary adaptation towards overcoming plant immunity, host barriers, and chemical stress such as fungicides, foreshadowing future outbreaks, host range shifts and expansions as well as potential pandemics by these pathogens., (© 2023 The Authors. Molecular Ecology published by John Wiley & Sons Ltd.)

Plant resistance (R) and pathogen avirulence (Avr) gene interactions play a vital role in pathogen resistance. Efficient molecular screening tools for crops lack far behind their model organism counterparts, yet they are essential to rapidly identify agriculturally important molecular interactions that trigger host resistance. Here, we have developed a novel wheat protoplast assay that enables efficient screening of Avr/R interactions at scale. Our assay allows access to the extensive gene pool of phenotypically described R genes because it does not require the overexpression of cloned R genes. It is suitable for multiplexed Avr screening, with interactions tested in pools of up to 50 Avr candidates. We identified Avr/R-induced defense genes to create a promoter-luciferase reporter. Then, we combined this with a dual-color ratiometric reporter system that normalizes read-outs accounting for experimental variability and Avr/R-induced cell death. Moreover, we introduced a self-replicative plasmid reducing the amount of plasmid used in the assay. Our assay increases the throughput of Avr candidate screening, accelerating the study of cellular defense signaling and resistance gene identification in wheat. We anticipate that our assay will significantly accelerate Avr identification for many wheat pathogens, leading to improved genome-guided pathogen surveillance and breeding of disease-resistant crops., (© 2024 The Authors. New Phytologist © 2024 New Phytologist Foundation.)

Crops are constantly exposed to pathogenic microbes. Rust fungi are examples of these harmful microorganisms, which have a major economic impact on wheat production. To protect themselves from pathogens like rust fungi, plants employ a multilayered immune system that includes immunoreceptors encoded by resistance genes. Significant efforts have led to the isolation of numerous resistance genes against rust fungi in cereals, especially in wheat. However, the evolution of virulence of rust fungi hinders the durability of resistance genes as a strategy for crop protection. Rust fungi, like other biotrophic pathogens, secrete an arsenal of effectors to facilitate infection, and these are the molecules that plant immunoreceptors target for pathogen recognition and mounting defense responses. When recognized, these effector proteins are referred to as avirulence (Avr) effectors. Despite the many predicted effectors in wheat rust fungi, only five Avr genes have been identified, all from wheat stem rust. Knowledge of the Avr genes and their variation in the fungal population will inform deployment of the most appropriate wheat disease-resistance genes for breeding and farming. The review provides an overview of methodologies as well as the validation techniques that have been used to characterize Avr effectors from wheat stem rust. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY 4.0 International license., Competing Interests: The author(s) declare no conflict of interest.

Net form net blotch (NFNB), caused by Pyrenophora teres f. teres , is an important barley disease. The centromeric region of barley chromosome 6H has often been associated with resistance or susceptibility to NFNB, including the broadly effective dominant resistance gene Rpt5 derived from barley line CIho 5791. We characterized a population of Moroccan P. teres f. teres isolates that had overcome Rpt5 resistance and identified quantitative trait loci (QTL) that were effective against these isolates. Eight Moroccan P. teres f. teres isolates were phenotyped on barley lines CIho 5791 and Tifang. Six isolates were virulent on CIho 5791, and two were avirulent. A CIho 5791 × Tifang recombinant inbred line (RIL) population was phenotyped with all eight isolates and confirmed the defeat of the 6H resistance locus formerly mapped as Rpt5 in barley line CI9819. A major QTL on chromosome 3H with the resistance allele derived from Tifang, as well as minor QTL, was identified and provided resistance against these isolates. F 2 segregation ratios supported dominant inheritance for both the 3H and 6H resistance. Furthermore, inoculation of progeny isolates derived from a cross of P. teres f. teres isolates 0-1 (virulent on Tifang/avirulent on CIho 5791) and MorSM 40-3 (avirulent on Tifang/virulent on CIho 5791) onto the RIL and F 2 populations determined that recombination between isolates can generate novel genotypes that overcome both resistance genes. Markers linked to the QTL identified in this study can be used to incorporate both resistance loci into elite barley cultivars for durable resistance., Competing Interests: The author(s) declare no conflict of interest.

Effectors encoded by avirulence genes (Avr) interact with the Phytophthora sojae resistance gene (Rps) products to generate incompatible interactions. The virulence profile of P. sojae is rapidly evolving as a result of the large-scale deployment of Rps genes in soybean. For a successful exploitation of Rps genes, it is recommended that soybean growers use cultivars containing the Rps genes corresponding to Avr genes present in P. sojae populations present in their fields. Determination of the virulence profile of P. sojae isolates is critical for the selection of soybean cultivars. High-resolution melting curve (HRM) analysis is a powerful tool, first applied in medicine, for detecting mutations with potential applications in different biological fields. Here, we report the development of an HRM protocol, as an original approach to discriminate effectors, to differentiate P. sojae haplotypes for six Avr genes. An HRM assay was performed on 24 P. sojae isolates with different haplotypes collected from soybean fields across Canada. The results clearly confirmed that the HRM assay discriminated different virulence genotypes. Moreover, the HRM assay was able to differentiate multiple haplotypes representing small allelic variations. HRM-based prediction was validated by phenotyping assays. This HRM assay provides a unique, cost-effective and efficient tool to predict virulence pathotypes associated with six different Avr (1b, 1c, 1d, 1k, 3a and 6) genes from P. sojae, which can be applied in the deployment of appropriate Rps genes in soybean fields., (© 2023 The Authors. Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

The deployment of disease resistance genes is currently the most economical and environmentally sustainable method of crop protection. However, disease resistance genes can rapidly break down because of constant pathogen evolution, particularly when they are deployed singularly. Polygenic resistance is, therefore, considered the most durable, but combining and maintaining these genes by breeding is a laborious process as effective genes are usually unlinked. The deployment of polygenic resistance with single-locus inheritance is a promising innovation that overcomes these difficulties while enhancing resistance durability. Because of major advances in genomic technologies, increasing numbers of plant resistance genes have been cloned, enabling the development of resistance transgene stacks (RTGSs) that encode multiple genes all located at a single genetic locus. Gene stacks encoding five stem rust resistance genes have now been developed in transgenic wheat and offer both breeding simplicity and potential resistance durability. The development of similar genomic resources in phytopathogens has advanced effector gene isolation and, in some instances, enabled functional validation of individual resistance genes in RTGS. Here, the wheat stem rust pathosystem is used as an illustrative example of how host and pathogen genomic advances have been instrumental in the development of RTGS, which is a strategy applicable to many other agricultural crop species., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Jost, Outram, Dibley, Zhang, Luo and Ayliffe.)

The gene-for-gene model proposed by H. H. Flor has been one of the fundamental precepts of plant-pathogen interactions that has underpinned decades of research towards our current concepts of plant immunity. The broad validity of this model as an elegant and accurate genetic description of specific recognition events between the products of plant resistance ( R ) and pathogen avirulence ( Avr ) genes has been demonstrated many times over in a wide variety of plant disease systems. In recent years detailed molecular and structural analyses have provided a deep understanding of the principles by which plant immune receptors recognize pathogen effectors, including providing molecular descriptions of many of the genetic loci in flax and flax rust characterized by Flor. Recent advances in molecular and structural understanding of immune receptor recognition and activation mechanisms have brought the field to a new level, where rational design of novel receptors through engineering approaches is becoming a realizable goal. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license., Competing Interests: The author(s) declare no conflict of interest.

Fungal effectors (small-secreted proteins) have long been considered as species or even subpopulation-specific. The increasing availability of high-quality fungal genomes and annotations has allowed the identification of trans-species or trans-genera families of effectors. Two avirulence effectors, AvrLm10A and AvrLm10B, of Leptosphaeria maculans, the fungus causing stem canker of oilseed rape, are members of such a large family of effectors. AvrLm10A and AvrLm10B are neighbouring genes, organized in divergent transcriptional orientation. Sequence searches within the L. maculans genome showed that AvrLm10A/AvrLm10B belong to a multigene family comprising five pairs of genes with a similar tail-to-tail organization. The two genes, in a pair, always had the same expression pattern and two expression profiles were distinguished, associated with the biotrophic colonization of cotyledons and/or petioles and stems. Of the two protein pairs further investigated, AvrLm10A_like1/AvrLm10B_like1 and AvrLm10A_like2/AvrLm10B_like2, the second one had the ability to physically interact, similarly to what was previously described for the AvrLm10A/AvrLm10B pair, and cross-interactions were also detected for two pairs. AvrLm10A homologues were identified in more than 30 Dothideomycete and Sordariomycete plant-pathogenic fungi. One of them, SIX5, is an effector from Fusarium oxysporum f. sp. lycopersici physically interacting with the avirulence effector Avr2. We found that AvrLm10A/SIX5 homologues were associated with at least eight distinct putative effector families, suggesting that AvrLm10A/SIX5 is able to cooperate with different effectors. These results point to a general role of the AvrLm10A/SIX5 proteins as "cooperating proteins", able to interact with diverse families of effectors whose encoding gene is co-regulated with the neighbouring AvrLm10A homologue., (© 2023 The Authors. Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

Plant effector biology is a research area that describes how plant-associated organisms modulate host structures and function to promote colonization by using small molecules (effectors). In this article, we analyzed 249 highly cited publications focused on plant pathogen effectors ( i.e. , Highly Influential studies on plant Pathogen Effectors; thereafter HIPEs) published between 2000 and 2020. This analysis identifies countries, organizations, and journals that contributed HIPEs, and reveals the evolution of research trends, model molecules, and model organisms over the last two decades. We notably show an increasing proportion of studies focused on effectors of biotrophic and hemibiotrophic fungi upon time. Our snapshot of the highly influential plant effector biology papers may help new comers in the field to gain an analytical understanding of this research area., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Louet, Duplessis, Frey and Petre.)

The host plays an essential role in parasite transmission. The viability of the host-parasite relationship depends upon development of immune resistance and the induction of disease tolerance. Here I propose that pathogen coevolution of avirulence factors promoting host disease tolerance is an essential feature of the parasitic lifestyle., Competing Interests: Declaration of interests The author declares no competing interests., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Coxiella burnetii is a Gram-negative, intracellular bacterium that causes the zoonosis Q fever. Among the many natural isolates of C. burnetii recovered from various sources, the Dugway group exhibits unique genetic characteristics, including the largest C. burnetii genomes. These strains were isolated during 1954-1958 from wild rodents from the Utah, USA desert. Despite retaining phase I lipopolysaccharide and the type 4B secretion system, two critical virulence factors, avirulence has been reported in a guinea pig infection model. Using guinea pig models, we evaluated the virulence, whole-cell vaccine (WCV) efficacy, and post-vaccination hypersensitivity (PVH) potential of a representative Dugway strain. Consistent with prior reports, Dugway appeared to be highly attenuated compared to a virulent strain. Indeed, Dugway-infected animals showed similarly low levels of fever, body weight loss, and splenomegaly like Nine Mile II-infected animals. When compared to a human Q fever vaccine, QVax ® , Dugway WCV exhibited analogous protection against a heterologous Nine Mile I challenge. PVH was investigated in a skin-testing model which revealed significantly decreased maximum erythema in Dugway Δ dot/icm WCV-skin-tested animals compared to that of QVax ® . These data provide insight into this unique bacterial strain and implicate its potential use as a mutated WCV candidate.

Little is known about the impact of host immunity on sexual reproduction in fungal pathogens. In particular, it is unclear whether crossing requires both sexual partners to infect living plant tissues. We addressed this issue in a three-year experiment investigating different scenarios of Zymoseptoria tritici crosses according to the virulence ('vir') or avirulence ('avr') of the parents against a qualitative resistance gene. Co-inoculations ('vir × vir', 'avr × vir', 'avr × avr') and single inoculations were performed on a wheat cultivar carrying the Stb16q resistance gene (Cellule) and a susceptible cultivar (Apache), in the greenhouse. We assessed the intensity of asexual reproduction by scoring disease severity, and the intensity of sexual reproduction by counting the ascospores discharged from wheat residues. As expected, disease severity was more intense on Cellule for 'vir × vir' co-inoculations than for 'avr × vir' co-inoculations, with no disease for 'avr × avr'. However, all types of co-inoculation yielded sexual offspring, whether or not the parental strains caused plant symptoms. Parenthood was confirmed by genotyping (SSR markers), and the occurrence of crosses between (co-)inoculated and exogenous strains (other strains from the experiment, or from far away) was determined. We showed that symptomatic asexual infection was not required for a strain to participate in sexual reproduction, and deduced from this result that avirulent strains could be maintained asymptomatically "on" or "in" leaf tissues of plants carrying the corresponding resistant gene for long enough to reproduce sexually. In two of the three years, the intensity of sexual reproduction did not differ between the three types of co-inoculation in Cellule, suggesting that crosses involving avirulent strains are not anecdotal. We discuss the possible mechanisms explaining the maintenance of avirulence in Z. tritici populations and the potential impact of particular resistance deployments such as cultivar mixtures for limiting resistance breakdown., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Ralstonia solanacearum causes devastating diseases in a wide range of economically important crops. It secretes a large number of virulence factors, also known as effectors, to promote its infection, and some of them are recognized when the host plant contains corresponding resistance genes. In this study we showed that a type III effector RipTPS from the avirulent R. solanacearum strain GMI1000 (RipTPS G ) specifically induced cell death in Nicotiana tabacum , but not in Nicotiana benthamiana , whereas the RipTPS homolog in the virulent strain CQPS-1 (RipTPS C ) induced cell death in neither N. tabacum nor N. benthamiana . These results indicated that RipTPS G is recognized in N. tabacum . Expression of RipTPS G induced upregulation of hypersensitive response (HR) -related genes in N. tabacum . The virulence of CQPS-1 was reduced when RipTPS G was genetically introduced into CQPS-1, further confirming that RipTPS G functions as an avirulence determinant. Protein sequence alignment indicated that there are only three amino acid polymorphisms between RipTPS G and RipTPS C . Site-directed mutagenesis analyses confirmed that the three amino acid residues are jointly required for the recognition of RipTPS G in N. tabacum . Expression of either RipTPS G or RipTPS C suppressed flg22-triggered reactive oxygen species (ROS) burst in N. benthamiana , suggesting that RipTPS contributes to pathogen virulence. Mutating the conserved residues in RipTPS's trehalose-phosphate synthase (TPS) domain did not block its HR induction and defense suppression activity, indicating that the TPS activity is not required for RipTPS's avirulence and virulence function., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 An, Chen, Xu, Ouyang, Cao, Wang, Liu and Zhang.)

Strategies for plant resistance gene deployment aim to preserve their durability to highly adaptable fungal pathogens. While the pyramiding of resistance genes is often proposed as an effective way to increase their durability, molecular mechanisms by which the pathogen can overcome the resistance also are important aspects to take into account. Here, we report a counterexample where pyramiding of two resistance genes of Brassica napus , Rlm3 and Rlm7 , matching the Leptosphaeria maculans avirulence genes AvrLm3 and AvrLm4-7 , respectively, favored the selection of double-virulent isolates. We previously demonstrated that the presence of a functional AvrLm4-7 gene in an isolate masks the Rlm3-AvrLm3 recognition. Rlm7 was massively deployed in France since 2004. L. maculans populations were surveyed on a large scale (>7,600 isolates) over a period of 20 years, and resistance gene deployment at the regional scale was determined. Mutations in isolates overcoming both resistance genes were analyzed. All data indicated that the simultaneous success of Rlm7 , the deployment of varieties pyramiding Rlm3 and Rlm7 , along with the decrease in areas cultivated with Rlm3 only, contributed to the success of virulent isolates toward Rlm7 , and more recently to both Rlm3 and Rlm7 . Experimental field assays proved that resistance gene alternation was a better strategy compared with pyramiding in this context. Our study also illustrated an unusually high sequence diversification of AvrLm3 and AvrLm4-7 under such a selection pressure, and identified a few regions of the AvrLm4-7 protein involved in both its recognition by Rlm7 and in its AvrLm3-Rlm3 masking ability. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Pseudomonas syringae is an evolutionarily diverse bacterial species complex and a preeminent model for the study of plant-pathogen interactions due in part to its remarkably broad host range. A critical feature of P. syringae virulence is the employment of suites of type III secreted effector (T3SE) proteins, which vary widely in composition and function. These effectors act on a variety of plant intracellular targets to promote pathogenesis but can also be avirulence factors when detected by host immune complexes. In this review, we survey the phylogenetic diversity (PD) of the P. syringae effectorome, comprising 70 distinct T3SE families identified to date, and highlight how avoidance of host immune detection has shaped effectorome diversity through functional redundancy, diversification, and horizontal transfer. We present emerging avenues for research and novel insights that can be gained via future investigations of plant-pathogen interactions through the fusion of large-scale interaction screens and phylogenomic approaches.

Pseudomonas savastanoi pv. phaseolicola is a bacterium that causes halo blight in beans. Different varieties of beans have hypersensitive resistance to specific races of P . savastanoi pv. phaseolicola . During hypersensitive resistance, also known as effector-triggered immunity (ETI), beans produce hormones that signal molecular processes to produce phytoalexins that are presumed to be antibiotic to bacteria. To shed light on hormone and phytoalexin production during immunity, we inoculated beans with virulent and avirulent races of P . savastanoi pv. phaseolicola . We then used mass spectrometry to measure the accumulation of salicylic acid (SA), the primary hormone that controls immunity in plants, and other hormones including jasmonate, methyljasmonate, indole-3-acetic acid, abscisic acid, cytokinin, gibberellic acid, and 1-aminocyclopropane-1-carboxylic acid. SA, but no other examined hormone, consistently increased at sites of infection to greater levels in resistant beans compared with susceptible beans at 4 days after inoculation. We then monitored 10 candidate bean phytoalexins. Daidzein, genistein, kievitone, phaseollin, phaseollidin, coumestrol, and resveratrol substantially increased alongside SA in resistant beans but not in susceptible beans. In vitro culture assays revealed that SA, daidzein, genistein, coumestrol, and resveratrol inhibited P . savastanoi pv. phaseolicola race 5 culture growth. These results demonstrate that these phytoalexins may be regulated by SA and work with SA during ETI to restrict bacterial replication. This is the first report of antibiotic activity for daidzein, genistein, and resveratrol to P . savastanoi pv. phaseolicola . These results improve our understanding of the mechanistic output of ETI toward this bacterial pathogen of beans.

One billion people worldwide are affected by fungal pathogens, of which 1.6 million succumb to fungal infections per year. This review discusses the emergence and evolution of fungal pathogenesis in humans in the form of opportunistic commensal and environmental fungi. We explore the attributes that contribute to their success as pathogens and the scenarios which may have caused the evolutionary selection of virulence factors. This includes antivirulence and avirulence genes, notions that are new for fungal pathogens of humans but which are based on well established concepts in bacterial pathogens and phytopathogenic fungi. These ideas will ultimately help us to better understand the pathogenicity of fungi that infect humans: from the emergence to the finer adjustment of virulence to promote pathogen persistence., Competing Interests: Declaration of interests No interests are declared., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Ascochyta lentis is a fungal pathogen that causes ascochyta blight in the important grain legume species lentil, but little is known about the molecular mechanism of disease or host specificity. We employed a map-based cloning approach using a biparental A. lentis population to clone the gene AlAvr1-1 that encodes avirulence towards the lentil cultivar PBA Hurricane XT. The mapping population was produced by mating A. lentis isolate P94-24, which is pathogenic on the cultivar Nipper and avirulent towards Hurricane, and the isolate AlKewell, which is pathogenic towards Hurricane but not Nipper. Using agroinfiltration, we found that AlAvr1-1 from the isolate P94-24 causes necrosis in Hurricane but not in Nipper. The homologous corresponding gene in AlKewell, AlAvr1-2, encodes a protein with amino acid variation at 23 sites and four of these sites have been positively selected in the P94-24 branch of the phylogeny. Loss of AlAvr1-1 in a gene knockout experiment produced a P94-24 mutant strain that is virulent on Hurricane. Deletion of AlAvr1-2 in AlKewell led to reduced pathogenicity on Hurricane, suggesting that the gene may contribute to disease in Hurricane. Deletion of AlAvr1-2 did not affect virulence for Nipper and AlAvr1-2 is therefore not an avirulence gene for Nipper. We conclude that the hemibiotrophic pathogen A. lentis has an avirulence effector, AlAvr1-1, that triggers a hypersensitive resistance response in Hurricane. This is the first avirulence gene to be characterized in a legume pathogen from the Pleosporales and may help progress research on other damaging Ascochyta pathogens., (© 2022 The Authors. Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

Septoria tritici blotch (STB), caused by the fungus Zymoseptoria tritici , is among the most threatening wheat diseases in Europe. Genetic resistance remains one of the main environmentally sustainable strategies to efficiently control STB. However, the molecular and physiological mechanisms underlying resistance are still unknown, limiting the implementation of knowledge-driven management strategies. Among the 22 known major resistance genes ( Stb ), the recently cloned Stb16q gene encodes a cysteine-rich receptor-like kinase conferring a full broad-spectrum resistance against Z. tritici . Here, we showed that an avirulent Z. tritici inoculated on Stb16q quasi near isogenic lines (NILs) either by infiltration into leaf tissues or by brush inoculation of wounded tissues partially bypasses Stb16q -mediated resistance. To understand this bypass, we monitored the infection of GFP-labeled avirulent and virulent isolates on Stb16q NILs, from germination to pycnidia formation. This quantitative cytological analysis revealed that 95% of the penetration attempts were unsuccessful in the Stb16q incompatible interaction, while almost all succeeded in compatible interactions. Infectious hyphae resulting from the few successful penetration events in the Stb16q incompatible interaction were arrested in the sub-stomatal cavity of the primary-infected stomata. These results indicate that Stb16q -mediated resistance mainly blocks the avirulent isolate during its stomatal penetration into wheat tissue. Analyses of stomatal aperture of the Stb16q NILs during infection revealed that Stb16q triggers a temporary stomatal closure in response to an avirulent isolate. Finally, we showed that infiltrating avirulent isolates into leaves of the Stb6 and Stb9 NILs also partially bypasses resistances, suggesting that arrest during stomatal penetration might be a common major mechanism for Stb -mediated resistances., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Battache, Lebrun, Sakai, Soudière, Cambon, Langin and Saintenac.)

Brassica napus (oilseed rape, canola) seedling resistance to Leptosphaeria maculans, the causal agent of blackleg (stem canker) disease, follows a gene-for-gene relationship. The avirulence genes AvrLmS and AvrLep2 were described to be perceived by the resistance genes RlmS and LepR2, respectively, present in B. napus 'Surpass 400'. Here we report cloning of AvrLmS and AvrLep2 using two independent methods. AvrLmS was cloned using combined in vitro crossing between avirulent and virulent isolates with sequencing of DNA bulks from avirulent or virulent progeny (bulked segregant sequencing). AvrLep2 was cloned using a biparental cross of avirulent and virulent L. maculans isolates and a classical map-based cloning approach. Taking these two approaches independently, we found that AvrLmS and AvrLep2 are the same gene. Complementation of virulent isolates with this gene confirmed its role in inducing resistance on Surpass 400, Topas-LepR2, and an RlmS-line. The gene, renamed AvrLmS-Lep2, encodes a small cysteine-rich protein of unknown function with an N-terminal secretory signal peptide, which is a common feature of the majority of effectors from extracellular fungal plant pathogens. The AvrLmS-Lep2/LepR2 interaction phenotype was found to vary from a typical hypersensitive response through intermediate resistance sometimes towards susceptibility, depending on the inoculation conditions. AvrLmS-Lep2 was nevertheless sufficient to significantly slow the systemic growth of the pathogen and reduce the stem lesion size on plant genotypes with LepR2, indicating the potential efficiency of this resistance to control the disease in the field., (© 2022 The Authors. Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

Knowledge of the evolutionary processes which govern pathogen recognition is critical to understanding durable disease resistance. We determined how Phytophthora infestans effector PiAVR2 is recognised by evolutionarily distinct resistance proteins R2 and Rpi-mcq1. We employed yeast two-hybrid, co-immunoprecipitation, virus-induced gene silencing, transient overexpression, and phosphatase activity assays to investigate the contributions of BSL phosphatases to R2- and Rpi-mcq1-mediated hypersensitive response (R2 HR and Rpi-mcq1 HR, respectively). Silencing PiAVR2 target BSL1 compromises R2 HR. Rpi-mcq1 HR is compromised only when BSL2 and BSL3 are silenced. BSL1 overexpression increases R2 HR and compromises Rpi-mcq1. However, overexpression of BSL2 or BSL3 enhances Rpi-mcq1 and compromises R2 HR. Okadaic acid, which inhibits BSL phosphatase activity, suppresses both recognition events. Moreover, expression of a BSL1 phosphatase-dead (PD) mutant suppresses R2 HR, whereas BSL2-PD and BSL3-PD mutants suppress Rpi-mcq1 HR. R2 interacts with BSL1 in the presence of PiAVR2, but not with BSL2 and BSL3, whereas no interactions were detected between Rpi-mcq1 and BSLs. Thus, BSL1 activity and association with R2 determine recognition of PiAVR2 by R2, whereas BSL2 and BSL3 mediate Rpi-mcq1 perception of PiAVR2. R2 and Rpi-mcq1 utilise distinct mechanisms to detect PiAVR2 based on association with different BSLs, highlighting central roles of these effector targets for both disease and disease resistance., (© 2021 The Authors. New Phytologist © 2021 New Phytologist Foundation.)

Ralstonia solanacearum causes bacterial wilt disease in solanaceous crops. Identification of avirulence type III-secreted effectors recognized by specific disease resistance proteins in host plant species is an important step toward developing durable resistance in crops. In the present study, we show that R. solanacearum effector RipJ functions as an avirulence determinant in Solanum pimpinellifolium LA2093. In all, 10 candidate avirulence effectors were shortlisted based on the effector repertoire comparison between avirulent Pe_9 and virulent Pe_1 strains. Infection assays with transgenic strain Pe_1 individually carrying a candidate avirulence effector from Pe_9 revealed that only RipJ elicits strong bacterial wilt resistance in S. pimpinellifolium LA2093. Furthermore, we identified that several RipJ natural variants do not induce bacterial wilt resistance in S. pimpinellifolium LA2093. RipJ belongs to the YopJ family of acetyltransferases. Our sequence analysis indicated the presence of partially conserved putative catalytic residues. Interestingly, the conserved amino acid residues in the acetyltransferase catalytic triad are not required for effector-triggered immunity. In addition, we show that RipJ does not autoacetylate its lysine residues. Our study reports the identification of the first R. solanacearum avirulence protein that triggers bacterial wilt resistance in tomato. We expect that our discovery of RipJ as an avirulence protein will accelerate the development of bacterial wilt-resistant tomato varieties in the future.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Pathogens and plants are in a constant battle with one another, the result of which is either the restriction of pathogen growth via constitutive or induced plant defense responses or the pathogen colonization of plant cells and tissues that cause disease. Elicitins are a group of highly conserved proteins produced by certain oomycete species, and their sterol binding ability is recognized as an important feature in sterol-auxotrophic oomycetes. Elicitins also orchestrate other aspects of the interactions of oomycetes with their plant hosts. The function of elicitins as avirulence or virulence factors is controversial and is dependent on the host species, and despite several decades of research, the function of these proteins remains elusive. We summarize here our current understanding of elicitins as either defense-promoting or defense-suppressing agents and propose that more recent approaches such as the use of 'omics' and gene editing can be used to unravel the role of elicitins in host-pathogen interactions. A better understanding of the role of elicitins is required and deciphering their role in host-pathogen interactions will expand the strategies that can be adopted to improve disease resistance and reduce crop losses.

Blackleg, caused by Leptosphaeria maculans , is a major disease of canola in Canada, Australia, and Europe. For effective deployment of resistant varieties and disease management, it is crucial to understand the population structure of L. maculans . In this study, we analyzed L. maculans isolates from commercial fields in western Canada from 2014 to 2016 for the presence and frequency of avirulence ( Avr ) genes. A total of 1,584 isolates were examined for the presence of Avr genes AvrLm1 , AvrLm2 , AvrLm3 , AvrLm4 , AvrLm6 , AvrLm7 , AvrLm9 , AvrLepR1 , AvrLepR2 , and AvrLmS via a set of differential host genotypes carrying known resistance genes and a PCR assay. Several Avr genes showed a higher frequency in the pathogen population, such as AvrLm6 and AvrLm7 , which were present in >90% of isolates, whereas AvrLm3 , AvrLm9 , and AvrLepR2 showed frequencies of <10%. A total of 189 races (different combinations of Avr genes) were detected, with Avr-2-4-6-7-S , Avr-1-4-6-7 , and Avr-2-4-6-7 as the three predominant races. When the effect of crop rotation was assessed, only a 3-year rotation showed a significantly higher frequency of AvrLm2 relative to shorter rotations. This study provides the information for producers to select effective canola varieties for blackleg management and for breeders to deploy new R genes in disease resistance breeding in western Canada.

Ralstonia solanacearum causes bacterial wilt disease in many plant species. Type III-secreted effectors (T3Es) play crucial roles in bacterial pathogenesis. However, some T3Es are recognized by corresponding disease resistance proteins and activate plant immunity. In this study, we identified the R. solanacearum T3E protein RipAZ1 (Ralstonia injected protein AZ1) as an avirulence determinant in the black nightshade species Solanum americanum. Based on the S. americanum accession-specific avirulence phenotype of R. solanacearum strain Pe&#95;26, 12 candidate avirulence T3Es were selected for further analysis. Among these candidates, only RipAZ1 induced a cell death response when transiently expressed in a bacterial wilt-resistant S. americanum accession. Furthermore, loss of ripAZ1 in the avirulent R. solanacearum strain Pe&#95;26 resulted in acquired virulence. Our analysis of the natural sequence and functional variation of RipAZ1 demonstrated that the naturally occurring C-terminal truncation results in loss of RipAZ1-triggered cell death. We also show that the 213 amino acid central region of RipAZ1 is sufficient to induce cell death in S. americanum. Finally, we show that RipAZ1 may activate defence in host cell cytoplasm. Taken together, our data indicate that the nucleocytoplasmic T3E RipAZ1 confers R. solanacearum avirulence in S. americanum. Few avirulence genes are known in vascular bacterial phytopathogens and ripAZ1 is the first one in R. solanacearum that is recognized in black nightshades. This work thus opens the way for the identification of disease resistance genes responsible for the specific recognition of RipAZ1, which can be a source of resistance against the devastating bacterial wilt disease., (© 2021 The Authors. Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

Wheat blast caused by the Triticum pathotype of Pyricularia oryzae poses a serious threat to wheat production in South America and Asia and is now becoming a pandemic disease. Here, we show that Rmg8, a promising wheat gene for resistance breeding, is suppressed by PWT4, an effector gene of P. oryzae, and in turn that the suppression is counteracted by Rwt4, a wheat gene recognizing PWT4. When PWT4 was introduced into a wheat blast isolate carrying AVR-Rmg8 (an avirulence gene corresponding to Rmg8), PWT4 suppressed wheat resistance conferred by Rmg8. PWT4 did not alter the expression of AVR-Rmg8, but higher expression of PWT4 led to more efficient suppression. This suppression was observed in rwt4 carriers, but not in Rwt4 carriers, indicating that it is counteracted by Rwt4. PWT4 was assumed to have been horizontally transferred from a weed-associated cryptic species, P. pennisetigena, to an Avena isolate of P. oryzae in Brazil. This implies a potential risk of the acquisition of PWT4 by the wheat blast fungus and the 'breakdown' of Rmg8. We suggest that Rmg8 should be introduced together with Rwt4 into a wheat cultivar when it is used for resistance breeding., (© 2020 The Authors New Phytologist © 2020 New Phytologist Trust.)

Attempted infections of plants with fungi result in diverse outcomes ranging from symptom-less resistance to severe disease and even death of infected plants. The deleterious effect on crop yield have led to intense focus on the cellular and molecular mechanisms that explain the difference between resistance and susceptibility. This research has uncovered plant resistance or susceptibility genes that explain either dominant or recessive inheritance of plant resistance with many of them coding for receptors that recognize pathogen invasion. Approaches based on cell biology and phytochemistry have contributed to identifying factors that halt an invading fungal pathogen from further invasion into or between plant cells. Plant chemical defence compounds, antifungal proteins and structural reinforcement of cell walls appear to slow down fungal growth or even prevent fungal penetration in resistant plants. Additionally, the hypersensitive response, in which a few cells undergo a strong local immune reaction, including programmed cell death at the site of infection, stops in particular biotrophic fungi from spreading into surrounding tissue. In this review, we give a general overview of plant recognition and defence of fungal parasites tracing back to the early 20th century with a special focus on Triticeae and on the progress that was made in the last 30 years., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

Pseudomonas syringae pv. actinidiae ICMP 18884 biovar 3 (Psa3) produces necrotic lesions during infection of its kiwifruit host. Bacterial growth in planta and lesion formation are dependent upon a functional type III secretion system (T3S), which translocates multiple effector proteins into host cells. Associated with the T3S locus is the conserved effector locus (CEL), which has been characterized and shown to be essential for the full virulence in other P. syringae pathovars. Two effectors at the CEL, hopM1 and avrE1, as well as an avrE1-related non-CEL effector, hopR1, have been shown to be redundant in the model pathogen P. syringae pv. tomato DC3000 (Pto), a close relative of Psa. However, it is not known whether CEL-related effectors are required for Psa pathogenicity. The Psa3 allele of hopM1, and its associated chaperone, shcM, have diverged significantly from their orthologs in Pto. Furthermore, the CEL effector hopAA1-1, as well as a related non-CEL effector, hopAA1-2, have both been pseudogenized. We have shown that HopM1 does not contribute to Psa3 virulence due to a truncation in shcM, a truncation conserved in the Psa lineage, probably due to the need to evade HopM1-triggered immunity in kiwifruit. We characterized the virulence contribution of CEL and related effectors in Psa3 and found that only avrE1 and hopR1, additively, are required for in planta growth and lesion production. This is unlike the redundancy described for these effectors in Pto and indicates that these two Psa3 genes are key determinants essential for kiwifruit bacterial canker disease., (© 2020 The Authors. Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

Wheat stem rust caused by the fungus Puccinia graminis f. sp. tritici ( Pgt ), is regaining prominence due to the recent emergence of virulent isolates and epidemics in Africa, Europe and Central Asia. The development and deployment of wheat cultivars with multiple stem rust resistance ( Sr ) genes stacked together will provide durable resistance. However, certain disease resistance genes can suppress each other or fail in particular genetic backgrounds. Therefore, the function of each Sr gene must be confirmed after incorporation into an Sr -gene stack. This is difficult when using pathogen disease assays due to epistasis from recognition of multiple avirulence (Avr) effectors. Heterologous delivery of single Avr effectors can circumvent this limitation, but this strategy is currently limited by the paucity of cloned Pgt Avrs . To accelerate Avr gene cloning, we outline a procedure to develop a mutant population of Pgt spores and select for gain-of-virulence mutants. We used ethyl methanesulphonate (EMS) to mutagenize urediniospores and create a library of > 10,000 independent mutant isolates that were combined into 16 bulks of ~658 pustules each. We sequenced random mutants and determined the average mutation density to be 1 single nucleotide variant (SNV) per 258 kb. From this, we calculated that a minimum of three independently derived gain-of-virulence mutants is required to identify a given Avr gene. We inoculated the mutant library onto plants containing Sr43 , Sr44 , or Sr45 and obtained 9, 4, and 14 mutants with virulence toward Sr43 , Sr44 , or Sr45 , respectively. However, only mutants identified on Sr43 and Sr45 maintained their virulence when reinolculated onto the lines from which they were identified. We further characterized 8 mutants with virulence toward Sr43 . These also maintained their virulence profile on the stem rust international differential set containing 20 Sr genes, indicating that they were most likely not accidental contaminants. In conclusion, our method allows selecting for virulent mutants toward targeted resistance ( R ) genes. The development of a mutant library from as little as 320 mg spores creates a resource that enables screening against several R genes without the need for multiple rounds of spore multiplication and mutagenesis., (Copyright © 2020 Kangara, Kurowski, Radhakrishnan, Ghosh, Cook, Yu, Arora, Steffenson, Figueroa, Mohareb, Saunders and Wulff.)

Halo blight disease of beans is caused by a gram-negative bacterium, Pseudomonas syringae pv. phaseolicola . The disease is prevalent in South America and Africa and causes crop loss for indigent people who rely on beans as a primary source of daily nutrition. In susceptible beans, P. syringae pv. phaseolicola causes water-soaking at the site of infection and produces phaseolotoxin, an inhibitor of bean arginine biosynthesis. In resistant beans, P. syringae pv. phaseolicola triggers a hypersensitive response that limits the spread of infection. Here, we used high-throughput mass spectrometry to interrogate the responses to two different P. syringae pv. phaseolicola isolates on a single line of common bean, Phaseolus vulgaris PI G19833, with a reference genome sequence. We obtained quantitative information for 4,135 bean proteins. A subset of 160 proteins with similar accumulation changes during both susceptible and resistant reactions included salicylic acid responders EDS1 and NDR1, ethylene and jasmonic acid biosynthesis enzymes, and proteins enabling vesicle secretion. These proteins revealed the activation of a basal defense involving hormonal responses and the mobilization of extracellular proteins. A subset of 29 proteins specific to hypersensitive immunity included SOBIR1, a G-type lectin receptor-like kinase, and enzymes needed for glucoside and phytoalexin production. Virus-induced gene silencing revealed that the G-type lectin receptor-like kinase suppresses bacterial infection. Together, the results define the proteomics of disease resistance to P. syringae pv. phaseolicola in beans and support a model whereby the induction of hypersensitive immunity reinstates defenses targeted by P. syringae pv. phaseolicola .

Puccinia striiformis f. sp. tritici , the causal agent of wheat stripe (yellow) rust, is an obligate, biotrophic fungus. It was difficult to study the genetics of the pathogen due to the lack of sexual reproduction. The recent discovery of alternate hosts for P. striiformis f. sp. tritici makes it possible to study inheritance and map genes involved in its interaction with plant hosts. To identify avirulence ( Avr ) genes in P. striiformis f. sp. tritici , we developed a segregating population by selfing isolate 12-368 on barberry ( Berberis vulgaris ) plants under controlled conditions. The dikaryotic sexual population segregated for avirulent/virulent phenotypes on nine Yr single-gene lines. The parental and progeny isolates were whole-genome sequenced at >30× coverage using Illumina HiSeq PE150 technology. A total of 2,637 high-quality markers were discovered by mapping the whole-genome sequencing (WGS) reads to the reference genome of strain 93-210 and used to construct a genetic map, consisting of 41 linkage groups, spanning 7,715.0 centimorgans (cM) and covering 68 Mb of the reference genome. The recombination rate was estimated to be 1.81 ± 2.32 cM/10 kb. Quantitative trait locus analysis mapped six Avr gene loci to the genetic map, including an Avr cluster harboring four Avr genes, AvYr7 , AvYr43 , AvYr44 , and AvYrExp2 Aligning the genetic map to the reference genome identified Avr candidates and narrowed them to a small genomic region (<200 kb). The discovery of the Avr gene cluster is useful for understanding pathogen evolution, and the identification of candidate genes is an important step toward cloning Avr genes for studying molecular mechanisms of pathogen-host interactions. IMPORTANCE Stripe rust is a destructive disease of wheat worldwide. Growing resistant cultivars is the most effective, easy-to-use, economical, and environmentally friendly strategy for the control of the disease. However, P. striiformis f. sp. tritici can produce new virulent races that may circumvent race-specific resistance. Therefore, understanding the genetic basis of the interactions between wheat genes for resistance and P. striiformis f. sp. tritici genes for avirulence is useful for improving cultivar resistance for more effective control of the disease. This study developed a high-quality map that facilitates genomic and genetic studies of important traits related to pathogen pathogenicity and adaptation to different environments and crop cultivars carrying different resistance genes. The information on avirulence/virulence genes identified in this study can be used for guiding breeding programs to select combinations of genes for developing new cultivars with effective resistance to mitigate this devastating disease.

Background: The stripe rust pathogen, Puccinia striiformis f. sp. tritici (Pst), threats world wheat production. Resistance to Pst is often overcome by pathogen virulence changes, but the mechanisms of variation are not clearly understood. To determine the role of mutation in Pst virulence changes, in previous studies 30 mutant isolates were developed from a least virulent isolate using ethyl methanesulfonate (EMS) mutagenesis and phenotyped for virulence changes. The progenitor isolate was sequenced, assembled and annotated for establishing a high-quality reference genome. In the present study, the 30 mutant isolates were sequenced and compared to the wide-type isolate to determine the genomic variation and identify candidates for avirulence (Avr) genes., Results: The sequence reads of the 30 mutant isolates were mapped to the wild-type reference genome to identify genomic changes. After selecting EMS preferred mutations, 264,630 and 118,913 single nucleotide polymorphism (SNP) sites and 89,078 and 72,513 Indels (Insertion/deletion) were detected among the 30 mutant isolates compared to the primary scaffolds and haplotigs of the wild-type isolate, respectively. Deleterious variants including SNPs and Indels occurred in 1866 genes. Genome wide association analysis identified 754 genes associated with avirulence phenotypes. A total of 62 genes were found significantly associated to 16 avirulence genes after selection through six criteria for putative effectors and degree of association, including 48 genes encoding secreted proteins (SPs) and 14 non-SP genes but with high levels of association (P ≤ 0.001) to avirulence phenotypes. Eight of the SP genes were identified as avirulence-associated effectors with high-confidence as they met five or six criteria used to determine effectors., Conclusions: Genome sequence comparison of the mutant isolates with the progenitor isolate unraveled a large number of mutation sites along the genome and identified high-confidence effector genes as candidates for avirulence genes in Pst. Since the avirulence gene candidates were identified from associated SNPs and Indels caused by artificial mutagenesis, these avirulence gene candidates are valuable resources for elucidating the mechanisms of the pathogen pathogenicity, and will be studied to determine their functions in the interactions between the wheat host and the Pst pathogen.

Opportunistic commensal and environmental fungi can cause superficial to systemic diseases in humans. But how did these pathogens adapt to infect us and how does host-pathogen co-evolution shape their virulence potential? During evolution toward pathogenicity, not only do microorganisms gain virulence genes, but they also tend to lose non-adaptive genes in the host niche. Additionally, virulence factors can become detrimental during infection when they trigger host recognition. The loss of non-adaptive genes as well as the loss of the virulence potential of genes by adaptations to the host has been investigated in pathogenic bacteria and phytopathogenic fungi, where they are known as antivirulence and avirulence genes, respectively. However, these concepts are nearly unknown in the field of pathogenic fungi of humans. We think that this unnecessarily limits our view of human-fungal interplay, and that much could be learned if we applied a similar framework to aspects of these interactions. In this review, we, therefore, define and adapt the concepts of antivirulence and avirulence genes for human pathogenic fungi. We provide examples for analogies to antivirulence genes of bacterial pathogens and to avirulence genes of phytopathogenic fungi. Introducing these terms to the field of pathogenic fungi of humans can help to better comprehend the emergence and evolution of fungal virulence and disease.

Magnaporthe oryzae infects rice, wheat, and many grass species in the Poaceae family by secreting protein effectors. Here, we analyzed the distribution, sequence variation, and genomic context of effector candidate ( EFC ) genes in 31 isolates that represent five pathotypes of M. oryzae , three isolates of M. grisea , a sister species of M. oryzae , and one strain each for eight species in the family Magnaporthaceae to investigate how the host range expansion of M. oryzae has likely affected the evolution of effectors. We used the EFC genes of M. oryzae strain 70-15, whose genome has served as a reference for many comparative genomics analyses, to identify their homologs in these strains. We also analyzed the previously characterized avirulence ( AVR ) genes and single-copy orthologous ( SCO ) genes in these strains, which showed that the EFC and AVR genes evolved faster than the SCO genes. The EFC and AVR repertoires among M. oryzae pathotypes varied widely probably because adaptation to individual hosts exerted different types of selection pressure. Repetitive DNA elements appeared to have caused the variation of some EFC genes. Lastly, we analyzed expression patterns of the AVR and EFC genes to test the hypothesis that such genes are preferentially expressed during host infection. This comprehensive dataset serves as a foundation for future studies on the genetic basis of the evolution and host specialization in M. oryzae ., (Copyright © 2019 Kim, Ko, Song, Choi, Kim, Jeon, Cheong, Kang and Lee.)

Background: Thermophilic bacilli in both aerobic or facultative anaerobic forms have been isolated for over a hundred years from different mesophilic or thermophilic environments as they are potential source of bioactive secondary metabolites. But the taxonomic resolution in the Bacillus genus at species or at strain level is very challenging for the insufficient divergence of the 16S rRNA genes. One such recurring problem is among Bacillus anthracis , B . cereus and B . thuringiensis . The disease-causing B. anthracis strains have their characteristic virulence factors coded in two well-known plasmids, namely pXO1 (toxin genes) and pXO2 (capsule genes)., Objective: The present study aimed at the molecular and genomic characterization of a recently reported thermophilic and environmental isolate of B. anthracis , strain PFAB2., Methods: We performed comparative genomics between the PFAB2 genome and different strains of B. anthracis , along with closely related B. cereus strains., Results: The pangenomic analysis suggests that the PFAB2 genome harbors no complete prophage genes. Cluster analysis of Bray-Kurtis similarity resemblance matrix revealed that gene content of PFAB2 is more closely related to other environmental strains of B. anthracis . The secretome analysis and the in vitro and in vivo pathogenesis experiments corroborate the avirulent phenotype of this strain. The most probable explanation for this phenotype is the apparent absence of plasmids harboring genes for capsule biosynthesis and toxins secretion in the draft genome. Additional features of PFAB2 are good spore-forming and germinating capabilities and rapid replication ability., Conclusion: The high replication rate in a wide range of temperatures and culture media, the non-pathogenicity, the good spore forming capability and its genomic similarity to the Ames strain together make PFAB2 an interesting model strain for the study of the pathogenic evolution of B . anthracis ., (© 2019 Bentham Science Publishers.)

A key component in the management of many diseases of crops is the use of plant disease resistance genes. However, the discovery and then sequence identification of these plant genes is challenging, whereas the characterization of the molecules that they recognize, the effector/avirulence products in pathogens, is often considerably more straight forward. Effectors are small proteins secreted by pathogens that can play major roles in modulating a plant's defense against attack. Effectors can be used to guide breeding of resistance genes, to trigger defense responses, and are part of integrated disease management strategies for crop protection. This review covers the role of effector-driven biotechnology in controlling plant diseases caused by fungi or oomycetes. Given that multi-billion dollar agriculture crops are based in some cases on plants recognizing just a handful of such effector proteins, there is considerable scope to use more fully effector proteins as a biotechnology resource in agriculture., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Interactions between Leptosphaeria maculans, causal agent of stem canker of oilseed rape, and its Brassica hosts are models of choice to explore the multiplicity of 'gene-for-gene' complementarities and how they diversified to increased complexity in the course of plant-pathogen co-evolution. Here, we support this postulate by investigating the AvrLm10 avirulence that induces a resistance response when recognized by the Brassica nigra resistance gene Rlm10. Using genome-assisted map-based cloning, we identified and cloned two AvrLm10 candidates as two genes in opposite transcriptional orientation located in a subtelomeric repeat-rich region of the genome. The AvrLm10 genes encode small secreted proteins and show expression profiles in planta similar to those of all L. maculans avirulence genes identified so far. Complementation and silencing assays indicated that both genes are necessary to trigger Rlm10 resistance. Three assays for protein-protein interactions showed that the two AvrLm10 proteins interact physically in vitro and in planta. Some avirulence genes are recognized by two distinct resistance genes and some avirulence genes hide the recognition specificities of another. Our L. maculans model illustrates an additional case where two genes located in opposite transcriptional orientation are necessary to induce resistance. Interestingly, orthologues exist for both L. maculans genes in other phytopathogenic species, with a similar genome organization, which may point to an important conserved effector function linked to heterodimerization of the two proteins., (© 2019 The Authors. New Phytologist © 2019 New Phytologist Trust.)

Toxoplasma gondii typically causes lifelong chronic infection and has been identified in a variety of intermediate and definitive hosts. Felids are capable of serving as both intermediate and definite hosts for T. gondii infection. However, there is no direct evidence to prove that servals are the intermediate host of T. gondii. In this study, T. gondii antibodies were detected in a serval by a modified agglutination test (titer, 1:200). Viable T. gondii was isolated from the striated muscles of the serval. This strain was further propagated in cell culture and designated as TgServalCHn1. Genetic characterization of DNA derived from cell culture was performed by RFLP-PCR of 10 markers, as well as polymorphic ROP5 and ROP18 genes. Results showed that this strain of T. gondii belonged to the genotype ToxoDB#20. The ROP5 allele 4 and ROP18 allele 3 suggested that this strain was avirulent, which was further supported by infection in mice. Encephalitis, immune organ necrosis and focal mononuclear cell infiltration in multiple organs were the main pathology characteristics observed in BALB/C mice infected with the TgServalCHn1 strain. To our knowledge, the present study is the first to report the isolation of T. gondii from a serval, which gives direct evidence for servals serving as an intermediate host of T. gondii. The genotyping results revealed the presence of genotype ToxoDB#20 in central China, enriching the scope of the distribution of T. gondii genotypes in Asia., (© 2019 Blackwell Verlag GmbH.)

In this paper we have characterized the lineage of two traits associated with the coat proteins (CPs) of the tombusvirids: Silencing suppression and HR elicitation in Nicotiana species. We considered that the tombusvirid CPs might collectively be considered an effector, with the CP of each CP-encoding species comprising a structural variant within the family. Thus, a phylogenetic analysis of the CP could provide insight into the evolution of a pathogen effector. The phylogeny of the CP of tombusvirids indicated that CP representatives of the family could be divided into four clades. In two separate clades the CP triggered a hypersensitive response (HR) in Nicotiana species of section Alatae but did not have silencing suppressor activity. In a third clade the CP had a silencing suppressor activity but did not have the capacity to trigger HR in Nicotiana species. In the fourth clade, the CP did not carry either function. Our analysis illustrates how structural changes that likely occurred in the CP effector of progenitors of the current genera led to either silencing suppressor activity, HR elicitation in select Nicotiana species, or neither trait.

The oomycete pathogens Phytophthora infestans and P. capsici cause significant crop losses world-wide, threatening food security. In each case, pathogenicity factors, called RXLR effectors, contribute to virulence. Some RXLRs are perceived by resistance proteins to trigger host immunity, but our understanding of the demographic processes and adaptive evolution of pathogen virulence remains poor. Here, we describe PenSeq, a highly efficient enrichment sequencing approach for genes encoding pathogenicity determinants which, as shown for the infamous potato blight pathogen Phytophthora infestans, make up < 1% of the entire genome. PenSeq facilitates the characterization of allelic diversity in pathogen effectors, enabling evolutionary and population genomic analyses of Phytophthora species. Furthermore, PenSeq enables the massively parallel identification of presence/absence variations and sequence polymorphisms in key pathogen genes, which is a prerequisite for the efficient deployment of host resistance genes. PenSeq represents a cost-effective alternative to whole-genome sequencing and addresses crucial limitations of current plant pathogen population studies, which are often based on selectively neutral markers and consequently have limited utility in the analysis of adaptive evolution. The approach can be adapted to diverse microbes and pathogens., (© 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.)

Puccinia striiformis f. sp. tritici (Pst) is an obligate biotrophic fungal pathogen causing stripe rust, one of the most important diseases of wheat worldwide. Mutation is considered as one of the major mechanisms causing virulence changes in the pathogen population, but experimental evidence is limited. To study the effect of mutation on pathogen variation, we developed 33 mutant isolates by treating urediniospores of Pst race PSTv-18, avirulent to all of the 18 Yr single-gene lines used to differentiate Pst races, with ethyl methanesulfonate (EMS). These isolates were characterized as 24 races, including 19 new races, through virulence testing on the set of 18 wheat Yr single-gene differential lines; and as 21 multi-locus genotypes with 19 simple sequence repeat and 48 single-nucleotide polymorphism markers. Most of the mutant isolates had more than one avirulence gene and more than one marker locus changed compared to the wild type isolate, indicating that EMS is able to cause mutations at multiple genome sites. The results showed that mutation can cause substantial changes in both avirulence and other genomic regions. The different frequencies of virulence among the mutant isolates suggested homozygous or heterozygous avirulence loci in the parental isolate, or relative ease of mutation at some avirulence loci. The results are useful for understanding evolutionary mechanisms of the important fungal pathogen.

The first bacterial and viral avirulence ( avr) genes were cloned in 1984. Although virus and bacterial avr genes were physically isolated in the same year, the questions associated with their characterization after discovery were very different, and these differences had a profound influence on the narrative of host-pathogen interactions for the past 30 years. Bacterial avr proteins were subsequently shown to suppress host defenses, leading to their reclassification as effectors, whereas research on viral avr proteins centered on their role in the viral infection cycle rather than their effect on host defenses. Recent studies that focus on the multifunctional nature of plant virus proteins have shown that some virus proteins are capable of suppression of the same host defenses as bacterial effectors. This is exemplified by the P6 protein of Cauliflower mosaic virus (CaMV), a multifunctional plant virus protein that facilitates several steps in the infection, including modulation of host defenses. This review highlights the modular structure and multifunctional nature of CaMV P6 and illustrates its similarities to other, well-established pathogen effectors.

Genome-wide analyses of the effector- and toxin-encoding genes were used to examine the phylogenetics and evolution of pathogenicity amongst diverse strains of Pseudomonas syringae causing bacterial canker of cherry (Prunus avium), including pathovars P. syringae pv morsprunorum (Psm) races 1 and 2, P. syringae pv syringae (Pss) and P. syringae pv avii. Phylogenetic analyses revealed Psm races and P. syringae pv avii clades were distinct and were each monophyletic, whereas cherry-pathogenic strains of Pss were interspersed amongst strains from other host species. A maximum likelihood approach was used to predict effectors associated with pathogenicity on cherry. Pss possesses a smaller repertoire of type III effectors but has more toxin biosynthesis clusters than Psm and P. syringae pv avii. Evolution of cherry pathogenicity was correlated with gain of genes such as hopAR1 and hopBB1 through putative phage transfer and horizontal transfer respectively. By contrast, loss of the avrPto/hopAB redundant effector group was observed in cherry-pathogenic clades. Ectopic expression of hopAB and hopC1 triggered the hypersensitive reaction in cherry leaves, confirming computational predictions. Cherry canker provides a fascinating example of convergent evolution of pathogenicity that is explained by the mix of effector and toxin repertoires acting on a common host., (© 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.)

Leptosphaeria maculans, the causal agent of blackleg disease, interacts with Brassica napus (oilseed rape, canola) and other Brassica hosts in a gene-for-gene manner. The avirulence gene AvrLmJ1 has been cloned previously and shown to interact with an unidentified Brassica juncea resistance gene. In this study, we show that the AvrLmJ1 gene maps to the same position as the AvrLm5 locus. Furthermore, isolates complemented with the AvrLmJ1 locus confer avirulence towards B. juncea genotypes harbouring Rlm5. These findings demonstrate that AvrLmJ1 is AvrLm5 and highlight the need for shared resources to characterize accurately avirulence and/or resistance genes., (© 2017 BSPP AND JOHN WILEY & SONS LTD.)

Recent advances in genomics and single-cell analysis have demonstrated the extraordinary complexity reached by microbial populations within their hosts. Communities range from complex multispecies groups to homogeneous populations differentiating into lineages through genetic or non-genetic mechanisms. Diversity within bacterial populations is recognized as a key driver of the evolution of animal pathogens. In plants, however, little is known about how interactions between different pathogenic and non-pathogenic variants within the host impact on defence responses, or how the presence within a mixture may affect the development or the fate of each variant. Using confocal fluorescence microscopy, we analysed the colonization of the plant apoplast by individual virulence variants of Pseudomonas syringae within mixed populations. We found that non-pathogenic variants can proliferate and even spread beyond the inoculated area to neighbouring tissues when in close proximity to pathogenic bacteria. The high bacterial concentrations reached at natural entry points promote such interactions during the infection process. We also found that a diversity of interactions take place at a cellular level between virulent and avirulent variants, ranging from dominant negative effects on proliferation of virulent bacteria to in trans suppression of defences triggered by avirulent bacteria. Our results illustrate the spatial dynamics and complexity of the interactions found within mixed infections, and their potential impact on pathogen evolution., (© 2017 BSPP AND JOHN WILEY & SONS LTD.)