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Volatiles play major roles in mediating ecological interactions between soil (micro)organisms and plants. It is well-established that microbial volatiles can increase root biomass and lateral root formation. To date, however, it is unknown whether microbial volatiles can affect directional root growth. Here, we present a novel method to study belowground volatile-mediated interactions. As proof-of-concept, we designed a root Y-tube olfactometer, and tested the effects of volatiles from four different soil-borne fungi on directional growth ofBrassica raparoots in soil. Subsequently, we compared the fungal volatile organic compounds (VOCs) previously profiled with Gas Chromatography-Mass Spectrometry (GC-MS). Using our newly designed setup, we show that directional root growth in soil is differentially affected by fungal volatiles. Roots grew more frequently toward volatiles from the root pathogenRhizoctonia solani, whereas volatiles from the other three saprophytic fungi did not impact directional root growth. GC-MS profiling showed that six VOCs were exclusively emitted byR. solani.These findings verify that this novel method is suitable to unravel the intriguing chemical cross-talk between roots and soil-borne fungi and its impact on root growth., Plant, Cell & Environment, 44 (1), ISSN:0140-7791, ISSN:1365-3040

1. Soil is a diverse and heterogeneous environment where chemicals mediate numerous interactions between soil organisms and plants. To date, studies have extensively addressed volatile-mediated interactions between soil microorganisms and the effects of microbial volatiles on plant growth. Yet, to our knowledge, it remains to be explored whether volatiles from soil-borne fungi can influence plant interactions with root herbivores, facilitating or hampering performance of competitors that share the same host plant. 2. In the present study, we investigated the effects of volatiles emitted by the soil-borne fungus Fusarium oxysporum on the performance of two root herbivores: the plant parasitic cyst nematode, Heterodera schachtii, and the insect root herbivore, Delia radicum, upon infestation of Brassica rapa roots. 3. Fungal volatiles slowed down the development of the root nematode cysts but increased their size, suggesting an enhanced egg load. In contrast, the performance of the insect root herbivore was unaffected by the exposure of roots to fungal volatiles. Additionally, fungal volatiles promoted the growth of plants infested with the root nematode, but not of those infested with the insect root herbivore. 4. Together, our data show that volatiles from a soil-borne fungus can affect root interactions with root herbivores. Increased production of nematode eggs and plant growth promotion suggest a specific modulation of root-herbivore interactions by fungal volatiles.

Microbiomes play a pivotal role in plant growth and health, but the genetic factors involved in microbiome assembly remain largely elusive. Here, 16S amplicon and metagenomic features of the rhizosphere microbiome were mapped as quantitative traits of a recombinant inbred line population of a cross between wild and domesticated tomato. Gene content analysis of prioritized tomato QTLs suggested a genetic basis for differential recruitment of various rhizobacterial lineages, including a Streptomyces-associated 6.31-Mbp region harboring tomato domestication sweeps and encoding, among others, the iron regulator FIT and the aquaporin SlTIP2.3. Within metagenome-assembled genomes of the rhizobacterial lineages Streptomyces and Cellvibrio, we identified microbial genes involved in metabolism of plant polysaccharides, iron, sulfur, trehalose, and vitamins, whose genetic variation associated with either modern or wild tomato QTLs. Integrating ‘microbiomics’ and quantitative plant genetics pinpointed putative plant and reciprocal microbial traits underlying microbiome assembly, thereby providing the first step towards plant-microbiome breeding programs.

The phyllosphere (i.e., the aerial parts of plants) harbors a rich microbial life, including bacteria, fungi, viruses, and yeasts. Current knowledge of yeasts stems primarily from industrial and medical research on Saccharomyces cerevisiae and Candida albicans, both of which can be found on plant tissues. For most other yeasts found in the phyllosphere, little is known about their ecology and functions. Here, we explore the diversity, dynamics, interactions, and genomics of yeasts associated with plant leaves and how tools and approaches developed for model yeasts can be adopted to disentangle the ecology and natural functions of phyllosphere yeasts. A first genomic survey exemplifies that we have only scratched the surface of the largely unexplored functional potential of phyllosphere yeasts.

Microorganisms colonizing plant surfaces and internal tissues provide a number of life-support functions for their host. Despite increasing recognition of the vast functional capabilities of the plant microbiome, our understanding of the ecology and evolution of the taxonomically hyperdiverse microbial communities is limited. Here, we review current knowledge of plant genotypic and phenotypic traits as well as allogenic and autogenic factors that shape microbiome composition and functions. We give specific emphasis to the impact of plant domestication on microbiome assembly and how insights into microbiomes of wild plant relatives and native habitats can contribute to reinstate or enrich for microorganisms with beneficial effects on plant growth, development, and health. Finally, we introduce new concepts and perspectives in plant microbiome research, in particular how community ecology theory can provide a mechanistic framework to unravel the interplay of distinct ecological processes—i.e., selection, dispersal, drift, diversification—that structure the plant microbiome.

Ancient societies first attempted to domesticate wild plants for food production, which gave rise to present-day agriculture. Nowadays, more than a third of agricultural production is lost due to abiotic or biotic stress, such as drought, salinity, pests and diseases. Current predictions indicating an expanding world population until the end of the century, one of humanity's greatest challenges should be how to feeding the world in a sustainable way, as future increases in crop yields should be achieved with fewer input of fertilizers and pesticides. These challenges have raised awareness of the importance of the plant root microbiome to improve agricultural and horticultural practices. Plant roots are colonized by a surprising number of microorganisms, revealing in most cases an intimate symbiotic relationship which facilitates nutrient uptake and provides the host plant with higher resistance against attackers. In this context, plants can be seen as "superorganisms" that depend on their root microbiome for important functions. But the impact of plant domestication on the functional diversity and beneficial activities of this root microbiome is still largely unknown. Recent studies showed taxonomic differences in the root microbiome between wild relatives and modern cultivars, mainly in root architecture and root exudation. This leads to the hypothesis that present-day cultivars might have lost traits to recruit and activate host-specific beneficial root microbiota. Our research project investigates tomato species and native soils in the Andes to explore the taxonomic and functional diversity of their root microbiomes. Next generation sequencing and ‘omics technologies, combined with classic microbiological techniques are being used to obtain insight in the diversity of root-associated microbial communities of tomatoes. We hypothesize that wild tomatoes grown in their native soils harbour unique and higher frequencies of beneficial root microbiota, compared to modern cultivars.

Upon herbivory, plants emit specific herbivore-induced plant volatiles (HIPVs) that can attract natural enemies of the herbivore thus serving as indirect plant resistance. Not only insect herbivores, but microorganisms may also affect HIPV emission before or after plant colonisation, which in turn can affect behaviour of natural enemies of the herbivore. Yet, it remains elusive whether volatiles from microorganisms influence HIPV emission and indirect plant resistance. In this study, we investigated whether exposure of Brassica rapa roots to volatiles from soil-borne fungi influence HIPV emission and the recruitment of natural enemies of Pieris brassicae larvae. Using a two-compartment pot system, we performed greenhouse and common-garden experiments, and we profiled plant HIPV emission. We found that exposure of plant roots to fungal volatiles did not affect the number of P. brassicae larvae recollected from the plants, suggesting a neutral effect of the fungal volatiles on natural predation. Likewise, in a greenhouse, similar numbers of larvae were parasitised by Cotesia glomerata wasps on control plants as on fungal volatile-exposed plants. Additionally, chemical analysis of HIPV profiles revealed no qualitative and quantitative differences between control plants and fungal volatile-exposed plants that were both infested with P. brassicae larvae. Together, our data indicate that root exposure to fungal volatiles did not affect indirect plant resistance to an insect herbivore. These findings provide new insight into the influence of indirect plant resistance by fungal volatiles that are discussed together with the effects of fungal volatiles on direct plant resistance., Ecological entomology, 45 (5), ISSN:0307-6946, ISSN:1365-2311

Plants have evolved resistance traits that negatively affect attackers, and tolerance traits that sustain plant growth despite herbivore damage. These mechanisms often co-occur in a mixed-defence strategy, balancing resistance and tolerance. These plant defences can be enhanced upon interaction with soil micro-organisms. Here we investigated the effects of volatiles emitted by soil-borne fungi on plant defence to insect herbivory, and on plant phenology. We exposed roots of Brassica rapa plants to volatiles emitted by four soil-borne fungi. As a proxy of plant resistance, we assessed the performance of Pieris brassicae, a caterpillar feeding on leaves and inflorescences, and of Delia radicum, an insect root herbivore. As a proxy of plant tolerance, we compared growth of volatile-exposed plants challenged with or without insects. Additionally, we assessed the effects on plant phenology by recording bolting time and by counting the number of buds and flowers. Plant exposure to fungal volatiles differentially affected plant resistance to above- and below-ground herbivory. Performance of P. brassicae caterpillars differed between the fungal volatile-exposed plants but was variable between experimental batches. In contrast, the effects of fungal volatiles on D. radicum performance were predominantly negative, indicating an increased plant resistance. Despite root consumption by D. radicum, root dry weight remained unchanged in infested plants compared with uninfested ones, irrespectively of the volatile exposure, suggesting compensation for the tissue loss, sometimes at the cost of undamaged above-ground tissues. When B. rapa plants were attacked by P. brassicae caterpillars, only exposure to volatiles of some fungi led to compensation for the loss of above-ground tissues consumed by the caterpillars, which differed between leaves and inflorescences. Furthermore, bolting was accelerated in response to volatiles of some fungi, resulting in more buds and flowers, which suggests a potential enhancement of plant fitness. Our data show that fungal volatiles can modulate the mixed-defence strategies of B. rapa plants, balancing plant resistance and tolerance to above- and below-ground herbivory. These effects may be variable and were fungus specific. Ultimately, plant fitness may be enhanced upon root exposure to fungal volatiles. A free Plain Language Summary can be found within the Supporting Information of this article.

The Dothideomycete Pseudocercospora fijiensis, previously Mycosphaerella fijiensis, is the causal agent of black Sigatoka, one of the most destructive diseases of bananas and plantains. Disease management depends on fungicide applications, with a major contribution from sterol demethylation-inhibitors (DMIs). The continued use of DMIs places considerable selection pressure on natural P. fijiensis populations, enabling the selection of novel genotypes with reduced sensitivity. The hitherto explanatory mechanism for this reduced sensitivity was the presence of non-synonymous point mutations in the target gene Pfcyp51, encoding the sterol 14α-demethylase enzyme. Here, we demonstrate a second mechanism involved in DMI sensitivity of P. fijiensis. We identified a 19-bp element in the wild-type (wt) Pfcyp51 promoter that concatenates in strains with reduced DMI sensitivity. A polymerase chain reaction (PCR) assay identified up to six Pfcyp51 promoter repeats in four field populations of P. fijiensis in Costa Rica. We used transformation experiments to swap the wt promoter of a sensitive field isolate with a promoter from a strain with reduced DMI sensitivity that comprised multiple insertions. Comparative in vivo phenotyping showed a functional and proportional up-regulation of Pfcyp51, which consequently decreased DMI sensitivity. Our data demonstrate that point mutations in the Pfcyp51 coding domain, as well as promoter inserts, contribute to the reduced DMI sensitivity of P. fijiensis. These results provide new insights into the importance of the appropriate use of DMIs and the need for the discovery of new molecules for black Sigatoka management.

Plant microbiome assembly is a spatial and dynamic process driven by root exudates and influenced by soil type, plant developmental stage and genotype. Genotype-dependent microbiome assembly has been reported for different crop plant species. Despite the effect of plant genetics on microbiome assembly, the magnitude of host control over its root microbiome is relatively small or, for many plant species, still largely unknown. Here we cultivated modern and wild tomato genotypes for four successive cycles and showed that divergence in microbiome assembly between the two genotypes was significantly amplified over time. Also, we show that the composition of the rhizosphere microbiome of modern and wild plants became more dissimilar from the initial bulk soil and from each other. Co-occurrence analyses further identified amplicon sequence variants (ASVs) associated with early and late successions of the tomato rhizosphere microbiome. Among the members of the Late Successional Rhizosphere microbiome, we observed an enrichment of ASVs belonging to the genera Acidovorax, Massilia and Rhizobium in the wild tomato rhizosphere, whereas the modern tomato rhizosphere was enriched for an ASV belonging to the genus Pseudomonas. Collectively, our approach allowed us to study the dynamics of rhizosphere microbiome over successional cultivation as well as to categorize rhizobacterial taxa for their ability to form transient or long-term associations with their host plants.

Plants are ubiquitously exposed to a wide diversity of (micro)organisms, including mutualists and antagonists. Prior to direct contact, plants can perceive microbial organic and inorganic volatile compounds (hereafter: volatiles) from a distance that, in turn, may affect plant development and resistance. To date, however, the specificity of plant responses to volatiles emitted by pathogenic and non-pathogenic fungi and the ecological consequences of such responses remain largely elusive. We investigated whether Arabidopsis thaliana plants can differentiate between volatiles of pathogenic and non-pathogenic soil-borne fungi. We profiled volatile organic compounds (VOCs) and measured CO2 emission of 11 fungi. We assessed the main effects of fungal volatiles on plant development and insect resistance. Despite distinct differences in VOC profiles between the pathogenic and non-pathogenic fungi, plants did not discriminate, based on plant phenotypic responses, between pathogenic and non-pathogenic fungi. Overall, plant growth was promoted and flowering was accelerated upon exposure to fungal volatiles, irrespectively of fungal CO2 emission levels. In addition, plants became significantly more susceptible to a generalist insect leaf-chewing herbivore upon exposure to the volatiles of some of the fungi, demonstrating that a prior fungal volatile exposure can negatively affect plant resistance. These data indicate that plant development and resistance can be modulated in response to exposure to fungal volatiles., Oecologia, 190 (3), ISSN:0029-8549, ISSN:1432-1939

Protecting plants from the inside out Some soils show a remarkable ability to suppress disease caused by plant pathogens, an ability that is attributed to plant-associated microbiota. Carrión et al. investigated the role of endophytes, the intimate microbial community found within roots, in fungal disease suppression (see the Perspective by Tringe). The wilt fungus Rhizoctonia solani infects sugar beets, whereupon transcriptional analysis shows that several bacterial endophyte species activate biosynthetic gene clusters to cause disease suppression. These organisms produce antifungal effectors, including enzymes that can digest fungal cell walls, and secondary metabolites, including phenazines, polyketides, and siderophores, which may contribute to the antifungal phenotype. Science , this issue p. 606 ; see also p. 568

Disease-suppressive soils are ecosystems in which plants suffer less from root infections due to the activities of specific microbial consortia. The characteristics of soils suppressive to specific fungal root pathogens are comparable to those of adaptive immunity in animals, as reported by Raaijmakers and Mazzola (Science 352:1392–3, 2016), but the mechanisms and microbial species involved in the soil suppressiveness are largely unknown. Previous taxonomic and metatranscriptome analyses of a soil suppressive to the fungal root pathogen Rhizoctonia solani revealed that members of the Burkholderiaceae family were more abundant and more active in suppressive than in non-suppressive soils. Here, isolation, phylogeny, and soil bioassays revealed a significant disease-suppressive activity for representative isolates of Burkholderia pyrrocinia, Paraburkholderia caledonica, P. graminis, P. hospita, and P. terricola. In vitro antifungal activity was only observed for P. graminis. Comparative genomics and metabolite profiling further showed that the antifungal activity of P. graminis PHS1 was associated with the production of sulfurous volatile compounds encoded by genes not found in the other four genera. Site-directed mutagenesis of two of these genes, encoding a dimethyl sulfoxide reductase and a cysteine desulfurase, resulted in a loss of antifungal activity both in vitro and in situ. These results indicate that specific members of the Burkholderiaceae family contribute to soil suppressiveness via the production of sulfurous volatile compounds.

CfAvr4, a chitin-binding effector protein produced by the Dothideomycete tomato pathogen Cladosporium fulvum, protects the cell wall of this fungus against hydrolysis by secreted host chitinases during infection. However, in the presence of the Cf-4 immune receptor of tomato, CfAvr4 triggers a hypersensitive response (HR), which renders the pathogen avirulent. Recently, several orthologues of CfAvr4 have been identified from phylogenetically closely related species of Dothideomycete fungi. Of these, DsAvr4 from Dothistroma septosporum also triggers a Cf-4-dependent HR, but CaAvr4 and CbAvr4 from Cercospora apii and Cercospora beticola, respectively, do not. All, however, bind chitin. To identify the region(s) and specific amino acid residue(s) of CfAvr4 and DsAvr4 required to trigger a Cf-4-dependent HR, chimeric and mutant proteins, in which specific protein regions or single amino acid residues, respectively, were exchanged between CfAvr4 and CaAvr4 or DsAvr4 and CbAvr4, were tested for their ability to trigger an HR in Nicotiana benthamiana plants transgenic for the Cf-4 immune receptor gene. Based on this approach, a single region common to CfAvr4 and DsAvr4 was determined to carry a conserved proline residue necessary for the elicitation of this HR. In support of this result, a Cf-4-dependent HR was triggered by mutant CaAvr4 and CbAvr4 proteins carrying an arginine-to-proline substitution at this position. This study provides the first step in deciphering how Avr4 orthologues from different Dothideomycete fungi trigger a Cf-4-dependent HR.

Previously, we have determined the nonhost-mediated recognition of the MfAvr4 and MfEcp2 effector proteins from the banana pathogen Mycosphaerella fijiensis in tomato, by the cognate Cf-4 and Cf-Ecp2 resistance proteins, respectively. These two resistance proteins could thus mediate resistance against M. fijiensis if genetically transformed into banana (Musa spp.). However, disease resistance controlled by single dominant genes can be overcome by mutated effector alleles, whose products are not recognized by the cognate resistance proteins. Here, we surveyed the allelic variation within the MfAvr4, MfEcp2, MfEcp2-2 and MfEcp2-3 effector genes of M. fijiensis in a global population of the pathogen, and assayed its impact on recognition by the tomato Cf-4 and Cf-Ecp2 resistance proteins, respectively. We identified a large number of polymorphisms that could reflect a co-evolutionary arms race between host and pathogen. The analysis of nucleotide substitution patterns suggests that both positive selection and intragenic recombination have shaped the evolution of M. fijiensis effectors. Clear differences in allelic diversity were observed between strains originating from South-East Asia relative to strains from other banana-producing continents, consistent with the hypothesis that M. fijiensis originated in the Asian-Pacific region. Furthermore, transient co-expression of the MfAvr4 effector alleles and the tomato Cf-4 resistance gene, as well as of MfEcp2, MfEcp2-2 and MfEcp2-3 and the putative Cf-Ecp2 resistance gene, indicated that effector alleles able to overcome these resistance genes are already present in natural populations of the pathogen, thus questioning the durability of resistance that can be provided by these genes in the field.

Plants and microorganisms are constantly engaged in highly dynamic interactions both above- and belowground. Several of these interactions are mediated by volatile organic compounds (VOCs), small carbon-based compounds with high vapor pressure at ambient temperature. In the rhizosphere, VOCs have an advantage in intra- and interorganismal signaling since they can diffuse through soil pores over longer distances than other metabolites and are not dependent on water availability. The research described in this PhD thesis explored how beneficial and pathogenic microorganisms that live in the rhizosphere and endosphere modulate plant growth, development and resistance via the production of VOCs. In vitro and in vivo bioassays as well as different ‘omic’ approaches, such as volatomics, transcriptomics and genomics, were employed to investigate underlying mechanisms of VOC-mediated microbe-microbe and microbe-plant interactions. To investigate the diversity and functions of microbial VOCs, a disease-suppressive soil was used as the source of the VOC-producing microorganisms. Previous metagenomics studies reported Actinobacteria, in particular Streptomyces and Microbacterium species, as the most abundant bacterial genera found in a soil naturally suppressive to the fungal root pathogen Rhizoctonia solani. VOCs of several Streptomyces isolates inhibited hyphal growth of R. solani and in addition, promoted plant growth. Coupling the Streptomyces VOC profiles with their effects on fungal growth pinpointed methyl 2-methylpentanoate and 1,3,5-trichloro-2-methoxy benzene as antifungal VOCs. Also Microbacterium isolates showed VOC-mediated antifungal activity and plant growth promotion. VOC profiling of Microbacterium sp. EC8 revealed several sulfur-containing compounds and ketones such as dimethyl disulfide, trimethyl trisulfide and 3,3,6-trimethylhepta-1,5-dien-4-one (also known as Artemisia ketone). Genome analysis of strain EC8 revealed genes involved in sulfur metabolism. Resolving the role of the identified compounds and genes in VOC-mediated plant growth promotion and induced resistance will be subject of future studies. VOC-mediated chemical warfare underground has been proposed as a key mechanism of natural disease-suppressive soils. The results presented in this thesis indeed point in that direction. However, to more conclusively determine the role of the identified Actinobacterial VOCs in soil suppressiveness to R. solani, it will be important to demonstrate that the fungicidal VOCs are actually produced in situ at the right place and at sufficient concentrations to suppress plant infection by the pathogenic fungus. In agriculture, VOCs and VOC-producing microorganisms provide a potential alternative to the use of pesticides to protect plants and to improve crop production. In the past decades, several in vitro studies have described the effects of microbial VOCs on other (micro)organisms. However, little is still known on the potential of VOCs in large-scale agriculture and horticulture. The results described in this thesis show that VOCs from Microbacterium sp. EC8 stimulate the growth of Arabidopsis, lettuce and tomato, but do not control damping-off disease of lettuce caused by R. solani. Significant biomass increases were also observed for plants exposed only shortly to the bacterial VOCs prior to transplantation of the seedlings to soil. These results indicate that VOCs from strain EC8 can prime plants for growth promotion without direct contact and prolonged colonization. Furthermore, the induction of the plant growth-promoting effects appeared to be plant tissue specific. Root exposure to the bacterial VOCs led to a significant increase in plant biomass whereas shoot exposure did not result in significant biomass increase of lettuce and tomato seedlings. Genome-wide transcriptome analysis of Arabidopsis seedlings exposed to VOCs from this bacterium showed an up-regulation of genes involved in sulfur and nitrogen metabolism and in ethylene and jasmonic acid signaling. These results suggest that the blend of VOCs of strain EC8 favors, in part, the plant’s assimilation of sulfate and nitrogen, essential nutrients for plant growth, development and also resistance. Similar to beneficial microorganisms, plant pathogenic microorganisms have also evolved strategies to modulate growth and defense of their hosts. For instance, compounds secreted by pathogens may suppress or interfere with plant defense. In this thesis I show that R. solani produces an array of VOCs that promote growth, accelerate development, change VOC emission and reduce insect resistance of plants. Plant growth-promoting effects induced by the fungal VOCs were not transgenerational. Genome-wide transcriptome analysis of Arabidopsis seedlings revealed that exposure to fungal VOCs caused up-regulation of genes involved in auxin signaling, but down-regulation of genes involved in ethylene and jasmonic acid signaling. These findings suggest that this soil-borne pathogen uses VOCs to predispose plants for infection by stimulating lateral root formation and enhancing root biomass while suppressing defense mechanisms. Alternatively, upon perception of VOCs from soil-borne pathogens, plants may invest in root biomass while minimizing investments in defense, a trade-off that helps them to speed up growth and reproduction and to survive pathogen attack. In conclusion, the research presented in this thesis shows that both plants and microorganisms engage via VOCs in long-distance interactions and that beneficial and pathogenic soil microorganisms can alter plant physiology in different ways. Here, I provided a first step in identifying microbial genes involved in the regulation of biologically active VOCs as well as candidate plant genes involved in VOC perception and signal transduction. How plants sense and differentiate among VOCs from beneficial and pathogenic soil microorganisms will be an intriguing subject for future studies.

CfAvr4, a chitin‐binding effector protein produced by the Dothideomycete tomato pathogen Cladosporium fulvum, protects the cell wall of this fungus against hydrolysis by secreted host chitinases during infection. However, in the presence of the Cf‐4 immune receptor of tomato, CfAvr4 triggers a hypersensitive response (HR), which renders the pathogen avirulent. Recently, several orthologues of CfAvr4 have been identified from phylogenetically closely related species of Dothideomycete fungi. Of these, DsAvr4 from Dothistroma septosporum also triggers a Cf‐4‐dependent HR, but CaAvr4 and CbAvr4 from Cercospora apii and Cercospora beticola, respectively, do not. All, however, bind chitin. To identify the region(s) and specific amino acid residue(s) of CfAvr4 and DsAvr4 required to trigger a Cf‐4‐dependent HR, chimeric and mutant proteins, in which specific protein regions or single amino acid residues, respectively, were exchanged between CfAvr4 and CaAvr4 or DsAvr4 and CbAvr4, were tested for their ability to trigger an HR in Nicotiana benthamiana plants transgenic for the Cf‐4 immune receptor gene. Based on this approach, a single region common to CfAvr4 and DsAvr4 was determined to carry a conserved proline residue necessary for the elicitation of this HR. In support of this result, a Cf‐4‐dependent HR was triggered by mutant CaAvr4 and CbAvr4 proteins carrying an arginine‐to‐proline substitution at this position. This study provides the first step in deciphering how Avr4 orthologues from different Dothideomycete fungi trigger a Cf‐4‐dependent HR.