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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. [ABSTRACT FROM AUTHOR]

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. [Figure: 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.

The wheat fungal pathogens Puccinia graminis f. sp. tritici (Pgt), P. striiformis f. sp. tritici, and P. triticina, causing stem, stripe, and leaf rust, respectively, pose a threat to global wheat production. Genetic resistance in the form of resistance (R) genes provides the best protection, but rust fungal populations change frequently by mutating avirulence (AVR) effectors matching specific R genes, thereby defeating resistance. Hence, characterization of AVR effectors is needed to understand the evolution of pathogen populations, provide insights for extending the effectiveness of R genes, and yield tools for the identification and isolation of R genes. Functional characterization of Avr genes in rust fungi is challenging in these biotrophic pathogens that lack a reliable and efficient transformation system. Studies indicate that the recently engineered foxtail mosaic virus (FoMV) shows promise as an expression system in cereals. In this study, we utilized two confirmed AVR effectors from Pgt, AVRSr35 and AVRSr50, to assess the applicability of FoMV for investigating rust fungus Avr genes. We showed that vector FoMV PV101 carrying PgtAvrSr35 induced a hypersensitive response (HR) in wheat having the corresponding Sr35 resistance gene. However, when carrying PgtAvrSr50, no HR or even mild viral symptoms were seen in a wheat line having Sr50, as this particular wheat line was not susceptible to FoMV. Several wheat cultivars did not support FoMV replication. The results here show that FoMV PV101 is effective as an expression vector for studying rust fungi AVR effectors, but its applicability relies on the susceptibility of wheat cultivars to FoMV. [Figure: see text] Copyright © 2024 His Majesty the King in Right of Canada, as represented by the Minister of Agriculture and Agri-Food Canada. This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Summary: 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. [ABSTRACT FROM AUTHOR]

SUMMARY: 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. Significance Statement: The development of a robust high‐throughput functional screening assay for pathogen effectors will facilitate the study in plant‐pathogen interactions. We established a novel screening system using the TMV‐GFP vector, enabling the rapid identification of effectors that interfere with plant immunity by observing GFP fluorescence under a UV lamp within a few days. The TMV‐GFP system is a promising approach for high‐throughput screening of effectors from diverse pathogens. [ABSTRACT FROM AUTHOR]

Microbes can overcome the ability of plant resistance genes to confer protection against disease by mutating their corresponding avirulence genes. The fungus Leptosphaeria maculans causes blackleg disease on canola crops and subverts Brassica napus resistance genes through several DNA mutation mechanisms. One of these is repeat‐induced point (RIP) mutation, which can 'leak' into the avirulence genes from the adjacent repetitive sequences that the mutation process is targeting. Here, we identified populations of L. maculans in Australia that have extensive RIP mutations in the avirulence gene AvrLm2 and show that this has been triggered by a duplication of the gene and surrounding DNA that includes the distant (>55 kb in total) AvrLm6 gene. This finding provides another mechanism of mutation by which pathogens can overcome host resistance, and more broadly contributes to understanding the complex balance between gene duplication and genome defence. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [Graphic: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Summary: 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 x 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. F2 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 F2 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. [ABSTRACT FROM AUTHOR]

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. [Graphic: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Plant pathogens deliver effector proteins which enter plant cells in order to establish the disease. The resistance (R) proteins in the plants can recognize some of these effectors and initiate a race-specific resistance response. These effectors which aid in triggering defense signaling in host upon R-protein mediated recognition are termed as Avirulence (Avr) proteins. AvrPi54 is an Avr protein from an ascomycete fungus, Magnaporthe oryzae, which causes hypersensitive response in rice cells containing broad-spectrum R-gene, Pi54. In this study, we have analysed several characteristics of AvrPi54 protein computationally and studied its subcellular localization in presence and absence of Pi54 protein. Our results suggest AvrPi54 is a small hydrophobic protein with no significant homology with any known proteins available in any publicly available database, till date. Transient expression of eYFP fused AvrPi54 protein (AvrPi54-eYFP) in epidermis of onion bulb suggested its translocation to nucleus and plasma membrane. Further, Bimolecular fluorescence complementation (BiFC) assay in the tobacco leaf cells depicted that Pi54 and AvrPi54 proteins interact at plasma membrane. Based on the obtained evidences, we infer that AvrPi54 probably is functional in nucleus of rice cells, either as a transcription factor or as a chromatin remodeler, thus playing important role in immunocompromization of the host. However, presence of Pi54 protein in the host cell might restrict the trespass of AvrPi54 into the nucleus by interacting directly with it at the plasma membrane and thus eliciting defense response. [ABSTRACT FROM AUTHOR]

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.

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. [ABSTRACT FROM AUTHOR]

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 Δdot/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. [ABSTRACT FROM AUTHOR]

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 (RipTPSG) specifically induced cell death in Nicotiana tabacum, but not in Nicotiana benthamiana, whereas the RipTPS homolog in the virulent strain CQPS-1 (RipTPSC) induced cell death in neither N. tabacum nor N. benthamiana. These results indicated that RipTPSG is recognized in N. tabacum. Expression of RipTPSG induced upregulation of hypersensitive response (HR) -related genes in N. tabacum. The virulence of CQPS-1 was reduced when RipTPSG was genetically introduced into CQPS-1, further confirming that RipTPSG functions as an avirulence determinant. Protein sequence alignment indicated that there are only three amino acid polymorphisms between RipTPSG and RipTPSC. Site-directed mutagenesis analyses confirmed that the three amino acid residues are jointly required for the recognition of RipTPSG in N. tabacum. Expression of either RipTPSG or RipTPSC 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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.

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. Virulence factors of fungal pathogens of humans can emerge in environmental and commensal interactions but also evolve during colonization and infections of the host. Factors such as heat, resistance to phagocytosis or against antifungals can be explained by these interactions. Antivirulence and avirulence are well known phenomena in bacteria and fungal plant pathogens but are rarely considered in fungal pathogens of humans. In Candida albicans and related species, the peptide toxin candidalysin may be under selective pressure due to its virulence and avirulence properties. A loss of antivirulence functions may contribute to Cryptococcus neoformans infections and to Candida glabrata drug and phagocytosis resistance. Virulence factors of commensal fungi seem to have evolved toward prevention of excessive host damage in immunocompromised hosts (lower virulence) and immune recognition in immunocompetent hosts (lower avirulence). [ABSTRACT FROM AUTHOR]

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.[Graphic: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Ralstonia pseudosolanacearum (Rps), previously known as R. solanacearum phylotypes I and III is one of the causal agents of bacterial wilt, a devastating disease that affects more than 250 plant species. Emerging Rps strains were identified infecting new hosts. P824 Rps strain was isolated from blueberry in Florida. Rps strains including PD7123 were isolated from hybrid tea roses in several countries through Europe. P781 is a representative strain of Rps commonly found on mandevilla in Florida. UW757 is a strain isolated from osteospermum plants originating in Guatemala. These strains are phylogenetically closely related and of economic importance on their respective hosts. The objective of this study is to associate the Type 3 Effectors (T3Es) repertoire of these four strains with host specificity. Candidate T3E associated with host specificity to blueberry, tea rose, osteospermum, and mandevilla were identified by sequence homology. Pathogenicity assays on 8 hosts including, blueberry, mandevilla, osteospermum and tea rose with the 4 strains showed that both P824 and PD7123 are pathogenic to blueberry and tea rose. P781 is the only strain pathogenic to mandevilla and P824 is the only strain non-pathogenic to osteospermum. Hypotheses based on correlation of T3E presence/absence and pathogenicity profiles identified 3 candidate virulence and 3 avirulence T3E for host specificity to blueberry and tea rose. Two candidate avirulence T3E were identified for mandevilla, and one candidate virulence for osteospermum. The strategy applied here can be used to reduce the number of host specificity candidate genes in closely related strains with different hosts. [ABSTRACT FROM AUTHOR]

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.

Abstract 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.

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.

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.

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. [ABSTRACT FROM AUTHOR]

Summary: 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. [ABSTRACT FROM AUTHOR]

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.

The interaction between plants and pathogens is a very dynamic and complex relationship that also includes a high degree of specificity, and it is precisely this last characteristic which triggers such important responses in the survival of one or the other. The pathosystem formed by tomato (Solanum lycopersicum L.) and Fusarium oxysporum f. sp. lycopersici (Fol) has been the subject of multiple studies due to the importance of the vegetable worldwide and for the economic and ecological impact of the fungus responsible for the vascular wilt disease in tomato, causing losses that go up to 100%. One way to find alternatives for the management of any pathosystem is to know the actors involved and the mechanisms that govern the interaction through technological and scientific advances that clearly show how the interaction develops on a genetic level. This review collects the information from different scientific sources with focus on the knowledge of the fungus, tomato cultivation and plant defense applied to this pathosystem, as well as the molecular mechanisms.

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_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_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. [ABSTRACT FROM AUTHOR]

Summary: 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. [ABSTRACT FROM AUTHOR]

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.

ABSTRACT 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 (

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. [ABSTRACT FROM AUTHOR]

Leaf and stripe rust caused by Puccinia recondita and Puccinia striiformis are important diseases of wheat in Pakistan and worldwide. The survey was carried out to monitor virulence pattern among the pathogen populations on rust trap nurseries planted in four different locations of Pakistan in 2016 and 2017 years. The rust trap nurseries consisted of 39 leaf and 27 stripe rust near isogenic lines and data were recorded following modified Cobb's scale. No virulence was found in the leaf and stripe rust genes of Lr18, Lr19, Lr23+(GAZA), Lr28, Lr29, Lr32, Lr34, Lr35, Lr36 and Lr37 and Yr1, Yr3, Yr4, Yr5, Yr8, Yr9, Yr10, Yr15, Yr17, Yr18, Yr26, Yr28, Yr29, Opata, Yrcv, Yrsp, Super Kauz, PBW-343 and Aoc + Yra, respectively. Faisalabad-08, Punjab-11, AARI-2011, Millat-11, AAS-11, and Ujala-16 varieties exhibited effective resistance to both rust pathotypes. Moderately stripe rust resistance was noticed on Galaxy-13 ranging from 20–30 MRMS. Thus, the use of resistant genes and their effective combination with other genes had paramount importance to combat both leaf and stripe rust effectively. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

This study analysed the phenotypic and genotypic variation among 511 Pseudomonas savastanoi pv. phaseolicola (Psp) isolates, causing halo blight in mungbeans. Collected from symptomatic mungbean (Vigna radiata) crops throughout Australia between 2005 and 2018, a total of 352 Psp isolates were phenotypically screened. Our in planta screening against a set of four mungbean cultivars with known susceptible and resistant reactions revealed five distinctive pathotypes. Isolates belonging to pathotype 2 were the most prevalent at 84% and were found to be highly pathogenic towards all tested mungbean genotypes. Genomic variation was investigated for 205 isolates using DNA fingerprints, splitting the halo blight pathogen population into two broad genetic lineages. Further genetic testing for two known avirulence genes, avrPphE and avrPphF, identified the avrPphE gene in all the tested isolates and avrPphF present in all but two. To identify candidate avirulence genes unique to Psp isolates infecting mungbean in Australia, a comparative genomics study was undertaken on the whole-genome sequences of two epidemiologically important Psp isolates, T11544 and K4287. The information presented in this study has the potential to dramatically improve mungbean disease resistance now and into the future. [ABSTRACT FROM AUTHOR]

Recent advances in genomics have led to a greater understanding of blackleg disease of canola, caused by the fungus Leptosphaeria maculans. Genome sequences are available for several L. maculans isolates and different Brassica species, and several resistance and avirulence genes, which have a 'gene for gene' relationship, have been characterised. Although several pairs of complementary resistance and avirulence genes have been sequenced, the molecular basis of their recognition is unknown. Mechanisms responsible for conferring virulence include amino acid substitutions, gene deletion and Repeat Induced Point mutations of avirulence genes. In some cases, a single avirulence gene is recognised by two resistance genes, and virulence towards each resistance gene is conferred by separate mutations. Furthermore, some avirulence-resistance gene interactions are epistatic over others; for instance, the presence of AvrLm7 (avirulent towards Rlm7) masks the AvrLm3-Rlm3 interaction resulting in isolates phenotypically appearing virulent towards Rlm3 despite having the avirulent form of the AvrLm3 allele. Several high throughput methods, including the ascospore shower technique, are used to characterise cultivar specificity, frequency of avirulence genes, and to detect fungicide sensitivity of fungal populations from stubble. The frequency of virulence in field populations can change rapidly under selection pressure when cultivars with the same resistance genes are sown in subsequent seasons. If there is a high risk of breakdown of a particular source of resistance, canola growers can be advised to sow a different cultivar, thus avoiding an epidemic. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]