A robust high-throughput functional screening assay for plant pathogen effectors using the TMV-GFP vector.

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.

P3N-PIPO but not P3 is the avirulence determinant in melon carrying the Wmr resistance against watermelon mosaic virus, although they contain a common genetic determinant.

Viruses employ a series of diverse translational strategies to expand their coding capacity, which produces viral proteins with common domains and entangles virus-host interactions. P3N-PIPO, which is a transcriptional slippage product from the P3 cistron, is a potyviral protein dedicated to intercellular movement. Here, we show that P3N-PIPO from watermelon mosaic virus (WMV) triggers cell death when transiently expressed in Cucumis melo accession PI 414723 carrying the Wmr resistance gene. Surprisingly, expression of the P3N domain, shared by both P3N-PIPO and P3, can alone induce cell death, whereas expression of P3 fails to activate cell death in PI 414723. Confocal microscopy analysis revealed that P3N-PIPO targets plasmodesmata (PD) and P3N associates with PD, while P3 localizes in endoplasmic reticulum in melon cells. We also found that mutations in residues L35, L38, P41, and I43 of the P3N domain individually disrupt the cell death induced by P3N-PIPO, but do not affect the PD localization of P3N-PIPO. Furthermore, WMV mutants with L35A or I43A can systemically infect PI 414723 plants. These key residues guide us to discover some WMV isolates potentially breaking the Wmr resistance. Through searching the NCBI database, we discovered some WMV isolates with variations in these key sites, and one naturally occurring I43V variation enables WMV to systemically infect PI 414723 plants. Taken together, these results demonstrate that P3N-PIPO, but not P3, is the avirulence determinant recognized by Wmr, although the shared N terminal P3N domain can alone trigger cell death.IMPORTANCEThis work reveals a novel viral avirulence (Avr) gene recognized by a resistance (R) gene. This novel viral Avr gene is special because it is a transcriptional slippage product from another virus gene, which means that their encoding proteins share the common N-terminal domain but have distinct C-terminal domains. Amazingly, we found that it is the common N-terminal domain that determines the Avr-R recognition, but only one of the viral proteins can be recognized by the R protein to induce cell death. Next, we found that these two viral proteins target different subcellular compartments. In addition, we discovered some virus isolates with variations in the common N-terminal domain and one naturally occurring variation that enables the virus to overcome the resistance. These results show how viral proteins with common domains interact with a host resistance protein and provide new evidence for the arms race between plants and viruses.

Ancient variation of the AvrPm17 gene in powdery mildew limits the effectiveness of the introgressed rye Pm17 resistance gene in wheat.

Introgressions of chromosomal segments from related species into wheat are important sources of resistance against fungal diseases. The durability and effectiveness of introgressed resistance genes upon agricultural deployment is highly variable-a phenomenon that remains poorly understood, as the corresponding fungal avirulence genes are largely unknown. Until its breakdown, the Pm17 resistance gene introgressed from rye to wheat provided broad resistance against powdery mildew (Blumeria graminis). Here, we used quantitative trait locus (QTL) mapping to identify the corresponding wheat mildew avirulence effector AvrPm17. It is encoded by two paralogous genes that exhibit signatures of reoccurring gene conversion events and are members of a mildew sublineage specific effector cluster. Extensive haplovariant mining in wheat mildew and related sublineages identified several ancient virulent AvrPm17 variants that were present as standing genetic variation in wheat powdery mildew prior to the Pm17 introgression, thereby paving the way for the rapid breakdown of the Pm17 resistance. QTL mapping in mildew identified a second genetic component likely corresponding to an additional resistance gene present on the 1AL.1RS translocation carrying Pm17. This gene remained previously undetected due to suppressed recombination within the introgressed rye chromosomal segment. We conclude that the initial effectiveness of 1AL.1RS was based on simultaneous introgression of two genetically linked resistance genes. Our results demonstrate the relevance of pathogen-based genetic approaches to disentangling complex resistance loci in wheat. We propose that identification and monitoring of avirulence gene diversity in pathogen populations become an integral part of introgression breeding to ensure effective and durable resistance in wheat.

Secreted in Xylem 6 (SIX6) Mediates Fusarium oxysporum f. sp. fragariae Race 1 Avirulence on FW1-Resistant Strawberry Cultivars.

Fusarium oxysporum f. sp. fragariae (Fof) race 1 is avirulent on cultivars with the dominant resistance gene FW1, while Fof race 2 is virulent on FW1-resistant cultivars. We hypothesized there was a gene-for-gene interaction between a gene at the FW1 locus and an avirulence gene (AvrFW1) in Fof race 1. To identify a candidate AvrFW1, we compared genomes of 24 Fof race 1 and three Fof race 2 isolates. We found one candidate gene that was present in race 1, was absent in race 2, was highly expressed in planta, and was homologous to a known effector, secreted in xylem 6 (SIX6). We knocked out SIX6 in two Fof race 1 isolates by homologous recombination. All SIX6 knockout transformants (ΔSIX6) gained virulence on FW1/fw1 cultivars, whereas ectopic transformants and the wildtype isolates remained avirulent. ΔSIX6 isolates were quantitatively less virulent on FW1/fw1 cultivars Fronteras and San Andreas than fw1/fw1 cultivars. Seedlings from an FW1/fw1 × fw1/fw1 population were genotyped for FW1 and tested for susceptibility to a SIX6 knockout isolate. Results suggested that additional minor-effect quantitative resistance genes could be present at the FW1 locus. This work demonstrates that SIX6 acts as an avirulence factor interacting with a resistance gene at the FW1 locus. The identification of AvrFW1 enables surveillance for Fof race 2 and provides insight into the mechanisms of FW1-mediated resistance. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Comparative Analysis of the Avirulence Effectors Produced by the Fungal Stem Rust Pathogen of Wheat.

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.

Mutagenesis of Wheat Powdery Mildew Reveals a Single Gene Controlling Both NLR and Tandem Kinase-Mediated Immunity.

Blumeria graminis f. sp. tritici (Bgt) is a globally important fungal wheat pathogen. Some wheat genotypes contain powdery mildew resistance (Pm) genes encoding immune receptors that recognize specific fungal-secreted effector proteins, defined as avirulence (Avr) factors. Identifying Avr factors is vital for understanding the mechanisms, functioning, and durability of wheat resistance. Here, we present AvrXpose, an approach to identify Avr genes in Bgt by generating gain-of-virulence mutants on Pm genes. We first identified six Bgt mutants with gain of virulence on Pm3b and Pm3c. They all had point mutations, deletions or insertions of transposable elements within the corresponding AvrPm3b2/c2 gene or its promoter region. We further selected six mutants on Pm3a, aiming to identify the yet unknown AvrPm3a3 recognized by Pm3a, in addition to the previously described AvrPm3a2/f2. Surprisingly, Pm3a virulence in the obtained mutants was always accompanied by an additional gain of virulence on the unrelated tandem kinase resistance gene WTK4. No virulence toward 11 additional R genes tested was observed, indicating that the gain of virulence was specific for Pm3a and WTK4. Several independently obtained Pm3a-WTK4 mutants have mutations in Bgt-646, a gene encoding a putative, nonsecreted ankyrin repeat-containing protein. Gene expression analysis suggests that Bgt-646 regulates a subset of effector genes. We conclude that Bgt-646 is a common factor required for avirulence on both a specific nucleotide-binding leucine-rich repeat and a WTK immune receptor. Our findings suggest that, beyond effectors, another type of pathogen protein can control the race-specific interaction between powdery mildew and wheat. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Multiple deletions of candidate effector genes lead to the breakdown of partial grapevine resistance to downy mildew.

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.

Multiplexed effector screening for recognition by endogenous resistance genes using positive defense reporters in wheat protoplasts.

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.

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

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

A Moroccan Pyrenophora teres f. teres Population Defeats Rpt5, the Broadly Effective Resistance on Barley Chromosome 6H.

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. 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.

The broad use of the Pm8 resistance gene in wheat resulted in hypermutation of the AvrPm8 gene in the powdery mildew pathogen.

BACKGROUND: Worldwide wheat production is under constant threat by fast-evolving fungal pathogens. In the last decades, wheat breeding for disease resistance heavily relied on the introgression of chromosomal segments from related species as genetic sources of new resistance. The Pm8 resistance gene against the powdery mildew disease has been introgressed from rye into wheat as part of a large 1BL.1RS chromosomal translocation encompassing multiple disease resistance genes and yield components. Due to its high agronomic value, this translocation has seen continuous global use since the 1960s on large growth areas, even after Pm8 resistance was overcome by the powdery mildew pathogen. The long-term use of Pm8 at a global scale provided the unique opportunity to study the consequences of such extensive resistance gene application on pathogen evolution. RESULTS: Using genome-wide association studies in a population of wheat mildew isolates, we identified the avirulence effector AvrPm8 specifically recognized by Pm8. Haplovariant mining in a global mildew population covering all major wheat growing areas of the world revealed 17 virulent haplotypes of the AvrPm8 gene that grouped into two functional categories. The first one comprised amino acid polymorphisms at a single position along the AvrPm8 protein, which we confirmed to be crucial for the recognition by Pm8. The second category consisted of numerous destructive mutations to the AvrPm8 open reading frame such as disruptions of the start codon, gene truncations, gene deletions, and interference with mRNA splicing. With the exception of a single, likely ancient, gain-of-virulence mutation found in mildew isolates around the world, all AvrPm8 virulence haplotypes were found in geographically restricted regions, indicating that they occurred recently as a consequence of the frequent Pm8 use. CONCLUSIONS: In this study, we show that the broad and prolonged use of the Pm8 gene in wheat production worldwide resulted in a multitude of gain-of-virulence mechanisms affecting the AvrPm8 gene in the wheat powdery mildew pathogen. Based on our findings, we conclude that both standing genetic variation as well as locally occurring new mutations contributed to the global breakdown of the Pm8 resistance gene introgression.

Distribution of Three Verticillium dahliae Races in Coastal California and Evaluation of Resistance in Lettuce.

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.

A rapid, equipment-free method for detecting avirulence genes of Pyricularia oryzae using a lateral flow strip-based RPA assay.

Rice blast, caused by Pyricularia oryzae, is one of the most destructive rice diseases worldwide. Using resistant rice varieties is the most cost-effective way to control rice blast. Consequently, it is critical to monitor the distribution frequency of avirulence genes in rice planting field to facilitate the breedings of resistant rice varieties. In this study, we established a rapid RPA-LFD detection system for the identification of AvrPik, Avr-Piz-t and Avr-Pi9. The optimized reaction temperature and duration were 37°C and 20 min, indicating that the reaction system could be initiated by body temperature without relying on any precision instruments. Specificity analysis showed that the primer and probe combinations targeting three Avr genes exhibited a remarkable specificity for at genus-level detection. Under the optimized condition, the lower detected thresholds of AvrPik, Avr-Piz-t and Avr-Pi9 were 10 fg/µl, 100 fg/µl and 10 pg/µl, respectively. Notably, the detection sensitivity of three Avr genes was much higher than that of PCR. In addition, we also successfully detected the presence of AvrPik, Avr-Piz-t and Avr-Pi9 in the leaf and panicle blast lesions with the RPA-LFD detection system. In particular, the genomic DNA was extracted using the simpler PEG-NaOH rapid extraction method. In summary, we developed the RPA detection system for AvrPik, Avr-Pi9 and Avr-Piz-t, combined with the PEG-NaOH rapid DNA extraction method. The innovative approach achieved rapid, real-time and accurate detection of three Avr genes in the field, which is helpful to understand the distribution frequency of the three Avr genes in the field and provide theoretical reference for the scientific layout of rice resistant varieties.

Whole Genome Resource and Genetic Analysis of Magnaporthe oryzae from Two Field Isolates in Northeast China.

To screen candidate fungal genes that may relate to avirulence genes corresponding to the host resistance genes, we characterized two field isolates of Magnaporthe oryzae that cause rice blast disease, especially in northeast China, and performed whole-genome resequencing of these two isolates. The genome assembly and annotation data was submitted to the National Center for Biotechnology Information database. Our study unveils the predicted fungal effectors of two dominant M. oryzae isolates in northeast China, providing a resource for Avr genes to clone. [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.

From Gene-for-Gene to Resistosomes: Flor's Enduring Legacy.

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.

Loss of PWT7, Located on a Supernumerary Chromosome, Is Associated with Parasitic Specialization of Pyricularia oryzae on Wheat.

Pyricularia oryzae, a blast fungus of gramineous plants, is composed of various host genus-specific pathotypes. The avirulence of an Avena isolate on wheat is conditioned by PWT3 and PWT4. We isolated the third avirulence gene from the Avena isolate and designated it as PWT7. PWT7 was effective as an avirulence gene only at the seedling stage or on leaves. PWT7 homologs were widely distributed in a subpopulation of the Eleusine pathotype and the Lolium pathotype but completely absent in the Triticum pathotype (the wheat blast fungus). The PWT7 homolog found in the Eleusine pathotype was one of the five genes involved in its avirulence on wheat. A comparative analysis of distribution of PWT7 and the other two genes previously identified in the Eleusine pathotype suggested that, in the course of parasitic specialization toward the wheat blast fungus, a common ancestor of the Eleusine, Lolium, Avena, and Triticum pathotypes first lost PWT6, secondly PWT7, and, finally, the function of PWT3. PWT7 or its homologs were located on core chromosomes in Setaria and Eleusine isolates but on supernumerary chromosomes in Lolium and Avena isolates. This is an example of interchromosomal translocations of effector genes between core and supernumerary chromosomes. [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.

A highly polymorphic effector protein promotes fungal virulence through suppression of plant-associated Actinobacteria.

Plant pathogens secrete effector proteins to support host colonization through a wide range of molecular mechanisms, while plant immune systems evolved receptors to recognize effectors or their activities to mount immune responses to halt pathogens. Importantly, plants do not act as single organisms, but rather as holobionts that actively shape their microbiota as a determinant of health. The soil-borne fungal pathogen Verticillium dahliae was recently demonstrated to exploit the VdAve1 effector to manipulate the host microbiota to promote vascular wilt disease in the absence of the corresponding immune receptor Ve1. We identify a multiallelic V. dahliae gene displaying c. 65% sequence similarity to VdAve1, named VdAve1-like (VdAve1L), which shows extreme sequence variation, including alleles that encode dysfunctional proteins, indicative of selection pressure to overcome host recognition. We show that the orphan cell surface receptor Ve2, encoded at the Ve locus, does not recognize VdAve1L. Additionally, we demonstrate that the full-length variant VdAve1L2 possesses antimicrobial activity, like VdAve1, yet with a divergent activity spectrum, that is exploited by V. dahliae to mediate tomato colonization through the direct suppression of antagonistic Actinobacteria in the host microbiota. Our findings open up strategies for more targeted biocontrol against microbial plant pathogens.

Long-term and rapid evolution in powdery mildew fungi.

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.

Genome-Wide Association to Study the Host-Specificity Determinants of Xanthomonas perforans.

Xanthomonas perforans and X. euvesicatoria are the causal agents of bacterial spot disease of tomato and pepper, endemic to the Southeastern United States. Although very closely related, the two bacterial species differ in host specificity, where X. perforans is the dominant pathogen of tomato and X. euvesicatoria that of pepper. This is in part due to the activity of avirulence proteins that are secreted by X. perforans strains and elicit effector-triggered immunity in pepper leaves, thereby restricting pathogen growth. In recent years, the emergence of several pepper-pathogenic X. perforans lineages has revealed variability within the bacterial species to multiply and cause disease in pepper, even in the absence of avirulence gene activity. Here, we investigated the basal evolutionary processes underlying the host range of this species using multiple genome-wide association analyses. Surprisingly, we identified two novel gene candidates that were significantly associated with pepper-pathogenic X. perforans and X. euvesicatoria. Both candidates were predicted to be involved in the transport/acquisition of nutrients common to the plant cell wall or apoplast and included a TonB-dependent receptor, which was disrupted through independent mutations within the X. perforans lineage. The other included a symporter of protons/glutamate, gltP, enriched with pepper-associated mutations near the promoter and start codon of the gene. Functional analysis of these candidates revealed that only the TonB-dependent receptor had a minor effect on the symptom development and growth of X. perforans in pepper leaves, indicating that pathogenicity to this host might have evolved independently within the bacterial species and is likely a complex, multigenic trait.

Polymorphism of Avirulence Genes and Adaptation to Brassica Resistance Genes Is Gene-Dependent in the Phytopathogenic Fungus Leptosphaeria maculans.

The fungal phytopathogen Leptosphaeria maculans, which causes stem canker (blackleg) of rapeseed (Brassica napus), is mainly controlled worldwide by genetic resistance, which includes major resistance genes (Rlm). This model is one of those for which the highest number of avirulence genes (AvrLm) has been cloned. In many systems, including the L. maculans-B. napus interaction, intense use of resistance genes exerts strong selection pressure on the corresponding avirulent isolates, and the fungi may rapidly escape resistance through various molecular events which modify the avirulence genes. In the literature, the study of polymorphism at avirulence loci is often focused on single genes under selection pressure. In this study, we investigate allelic polymorphism at 11 avirulence loci in a French population of 89 L. maculans isolates collected on a trap cultivar in four geographic locations in the 2017-2018 cropping season. The corresponding Rlm genes have been (i) used for a long time, (ii) recently used, or (iii) unused in agricultural practice. The sequence data generated indicate an extreme diversity of situations. For example, genes submitted to an ancient selection may have either been deleted in populations (AvrLm1) or replaced by a single-nucleotide mutated virulent version (AvrLm2, AvrLm5-9). Genes that have never been under selection may either be nearly invariant (AvrLm6, AvrLm10A, AvrLm10B), exhibit rare deletions (AvrLm11, AvrLm14), or display a high diversity of alleles and isoforms (AvrLmS-Lep2). These data suggest that the evolutionary trajectory of avirulence/virulence alleles is gene-dependent and independent of selection pressure in L. maculans. [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.