Ronnie de Jonge

Conserved Fungal LysM Effector Ecp6 Prevents Chitin-Triggered Immunity in Plants

Fungal Defenses One of the major driving forces of evolution is the constant arms race between plants and animals and the microbial pathogens that infect them. The fungus Cladosporium fulvum causes leaf mold on tomato plants. One of the ways tomato plants sense infections by C. fulvum is by detecting chitin, a component of fungal cell walls. In response, the fungus has evolved strategies to evade detection. De Jonge et al. (p. 953) have now identified one such mechanism in C. fulvum, mediated by the effector protein Ecp6. Secreted Ecp6 is able to bind to chitin oligosaccharides that are released upon degradation of the fungal cell wall and sequester them so that they are not detected by tomato chitin receptors. Proteins with domain structure similar to Ecp6 are conserved throughout the fungal kingdom, which suggests that chitin sequestration may represent a general mechanism used by fungi to evade immune detection. A fungal protein binds to a host cell wall component to allow the fungus to escape immune responses. Multicellular organisms activate immunity upon recognition of pathogen-associated molecular patterns (PAMPs). Chitin is the major component of fungal cell walls, and chitin oligosaccharides act as PAMPs in plant and mammalian cells. Microbial pathogens deliver effector proteins to suppress PAMP-triggered host immunity and to establish infection. Here, we show that the LysM domain–containing effector protein Ecp6 of the fungal plant pathogen Cladosporium fulvum mediates virulence through perturbation of chitin-triggered host immunity. During infection, Ecp6 sequesters chitin oligosaccharides that are released from the cell walls of invading hyphae to prevent elicitation of host immunity. This may represent a common strategy of host immune suppression by fungal pathogens, because LysM effectors are widely conserved in the fungal kingdom.

Tomato immune receptor Ve1 recognizes effector of multiple fungal pathogens uncovered by genome and RNA sequencing

Fungal plant pathogens secrete effector molecules to establish disease on their hosts, and plants in turn use immune receptors to try to intercept these effectors. The tomato immune receptor Ve1 governs resistance to race 1 strains of the soil-borne vascular wilt fungi Verticillium dahliae and Verticillium albo-atrum, but the corresponding Verticillium effector remained unknown thus far. By high-throughput population genome sequencing, a single 50-Kb sequence stretch was identified that only occurs in race 1 strains, and subsequent transcriptome sequencing of Verticillium-infected Nicotiana benthamiana plants revealed only a single highly expressed ORF in this region, designated Ave1 (for Avirulence on Ve1 tomato). Functional analyses confirmed that Ave1 activates Ve1-mediated resistance and demonstrated that Ave1 markedly contributes to fungal virulence, not only on tomato but also on Arabidopsis. Interestingly, Ave1 is homologous to a widespread family of plant natriuretic peptides. Besides plants, homologous proteins were only found in the bacterial plant pathogen Xanthomonas axonopodis and the plant pathogenic fungi Colletotrichum higginsianum, Cercospora beticola, and Fusarium oxysporum f. sp. lycopersici. The distribution of Ave1 homologs, coincident with the presence of Ave1 within a flexible genomic region, strongly suggests that Verticillium acquired Ave1 from plants through horizontal gene transfer. Remarkably, by transient expression we show that also the Ave1 homologs from F. oxysporum and C. beticola can activate Ve1-mediated resistance. In line with this observation, Ve1 was found to mediate resistance toward F. oxysporum in tomato, showing that this immune receptor is involved in resistance against multiple fungal pathogens.

The Soil-Borne Supremacy.

The rhizosphere microbiome plays an important role in plant growth, nutrition and health. Recent research unearthed that plant genotype-dependent navigation of microbial community composition in the rhizosphere is associated with fitness consequences for the host plant, providing great promise for breeding soil-borne supremacy traits into future crops.

Disease-induced assemblage of a plant-beneficial bacterial consortium

Disease suppressive soils typically develop after a disease outbreak due to the subsequent assembly of protective microbiota in the rhizosphere. The role of the plant immune system in the assemblage of a protective rhizosphere microbiome is largely unknown. In this study, we demonstrate that Arabidopsis thaliana specifically promotes three bacterial species in the rhizosphere upon foliar defense activation by the downy mildew pathogen Hyaloperonospora arabidopsidis. The promoted bacteria were isolated and found to interact synergistically in biofilm formation in vitro. Although separately these bacteria did not affect the plant significantly, together they induced systemic resistance against downy mildew and promoted growth of the plant. Moreover, we show that the soil-mediated legacy of a primary population of downy mildew infected plants confers enhanced protection against this pathogen in a second population of plants growing in the same soil. Together our results indicate that plants can adjust their root microbiome upon pathogen infection and specifically recruit a group of disease resistance-inducing and growth-promoting beneficial microbes, therewith potentially maximizing the chance of survival of their offspring that will grow in the same soil.

Verticillium dahliae LysM effectors differentially contribute to virulence on plant hosts

Summary Chitin‐binding lysin motif (LysM) effectors contribute to the virulence of various plant‐pathogenic fungi that are causal agents of foliar diseases. Here, we report the LysM effectors of the soil‐borne fungal vascular wilt pathogen Verticillium dahliae. Comparative genomics revealed three core LysM effectors that are conserved in a collection of V. dahliae strains. Remarkably, and in contrast with the previously studied LysM effectors of other plant pathogens, no expression of core LysM effectors was monitored in planta in a taxonomically diverse panel of host plants. Moreover, targeted deletion of the individual LysM effector genes in V. dahliae strain JR2 did not compromise virulence in infections on Arabidopsis, tomato or Nicotiana benthamiana. Interestingly, an additional lineage‐specific LysM effector is encoded in the genome of V. dahliae strain VdLs17, but not in any other V. dahliae strain sequenced to date. Remarkably, this lineage‐specific effector is expressed in planta and contributes to the virulence of V. dahliae strain VdLs17 on tomato, but not on Arabidopsis or N. benthamiana. Functional analysis revealed that this LysM effector binds chitin, is able to suppress chitin‐induced immune responses and protects fungal hyphae against hydrolysis by plant hydrolytic enzymes. Thus, in contrast with the core LysM effectors of V. dahliae, this lineage‐specific LysM effector of strain VdLs17 contributes to virulence in planta.

The Soil-Borne Legacy

Plants greatly rely on their root microbiome for uptake of nutrients and protection against stresses. Recent studies have uncovered the involvement of plant stress responses in the assembly of plant-beneficial microbiomes. To facilitate durable crop production, deciphering the driving forces that shape the microbiome is crucial.

The heterothallic sugarbeet pathogen Cercospora beticola contains exon fragments of both MAT genes that are homogenized by concerted evolution.

Dothideomycetes is one of the most ecologically diverse and economically important classes of fungi. Sexual reproduction in this group is governed by mating type (MAT) genes at the MAT1 locus. Self-sterile (heterothallic) species contain one of two genes at MAT1 (MAT1-1-1 or MAT1-2-1) and only isolates of opposite mating type are sexually compatible. In contrast, self-fertile (homothallic) species contain both MAT genes at MAT1. Knowledge of the reproductive capacities of plant pathogens are of particular interest because recombining populations tend to be more difficult to manage in agricultural settings. In this study, we sequenced MAT1 in the heterothallic Dothideomycete fungus Cercospora beticola to gain insight into the reproductive capabilities of this important plant pathogen. In addition to the expected MAT gene at MAT1, each isolate contained fragments of both MAT1-1-1 and MAT1-2-1 at ostensibly random loci across the genome. When MAT fragments from each locus were manually assembled, they reconstituted MAT1-1-1 and MAT1-2-1 exons with high identity, suggesting a retroposition event occurred in a homothallic ancestor in which both MAT genes were fused. The genome sequences of related taxa revealed that MAT gene fragment pattern of Cercospora zeae-maydis was analogous to C. beticola. In contrast, the genome of more distantly related Mycosphaerella graminicola did not contain MAT fragments. Although fragments occurred in syntenic regions of the C. beticola and C. zeae-maydis genomes, each MAT fragment was more closely related to the intact MAT gene of the same species. Taken together, these data suggest MAT genes fragmented after divergence of M. graminicola from the remaining taxa, and concerted evolution functioned to homogenize MAT fragments and MAT genes in each species.

Fermentation assays reveal differences in sugar and (off-) flavor metabolism across different Brettanomyces bruxellensis strains

Abstract Brettanomyces (Dekkera) bruxellensis is an ascomycetous yeast of major importance in the food, beverage and biofuel industry. It has been isolated from various man‐made ecological niches that are typically characterized by harsh environmental conditions such as wine, beer, soft drink, etc. Recent comparative genomics studies revealed an immense intraspecific diversity, but it is still unclear whether this genetic diversity also leads to systematic differences in fermentation performance and (off‐)flavor production, and to what extent strains have evolved to match their ecological niche. Here, we present an evaluation of the fermentation properties of eight genetically diverse B. bruxellensis strains originating from beer, wine and soft drinks. We show that sugar consumption and aroma production during fermentation are determined by both the yeast strain and composition of the medium. Furthermore, our results indicate a strong niche adaptation of B. bruxellensis, most clearly for wine strains. For example, only strains originally isolated from wine were able to thrive well and produce the typical Brettanomyces‐related phenolic off‐flavors 4‐ethylguaiacol and 4‐ethylphenol when inoculated in red wine. Sulfite tolerance was found as a key factor explaining the observed differences in fermentation performance and off‐flavor production. Sequence analysis of genes related to phenolic off‐flavor production, however, revealed only marginal differences between the isolates tested, especially at the amino acid level. Altogether, our study provides novel insights in the Brettanomyces metabolism of flavor production, and is highly relevant for both the wine and beer industry.

Genome Sequence of a Lethal Strain of Xylem-Invading Verticillium nonalfalfae

ABSTRACT Verticillium nonalfalfae, a soilborne vascular phytopathogenic fungus, causes wilt disease in several crop species. Of great concern are outbreaks of highly aggressive V. nonalfalfae strains, which cause a devastating wilt disease in European hops. We report here the genome sequence and annotation of V. nonalfalfae strain T2, providing genomic information that will allow better understanding of the molecular mechanisms underlying the development of highly aggressive strains.

Ancient duplication and horizontal transfer of a toxin gene cluster reveals novel mechanisms in the cercosporin biosynthesis pathway

Cercospora species have a global distribution and are best known as the causal agents of leaf spot diseases of many crops. Cercospora leaf spot (CLS) is an economically devastating disease of sugar beet caused by C. beticola. The C. beticola genome encodes 63 biosynthetic gene clusters, including the cercosporin toxin biosynthesis (CTB) cluster. Studies spanning nearly 60 years have shown that cercosporin is photoactivated, critical for disease development, and toxic to most organisms except Cercospora spp. themselves, which exhibit cercosporin auto-resistance. We show that the CTB gene cluster has experienced an unprecedented number of duplications, losses, and horizontal transfers across a spectrum of plant pathogenic fungi. Although cercosporin biosynthesis has been widely assumed to rely on the eight gene CTB cluster, our comparative genomic analysis revealed extensive gene collinearity adjacent to the established cluster in all CTB cluster-harboring species. We demonstrate that the CTB cluster is larger than previously recognized and includes the extracellular proteins fasciclin and laccase required for cercosporin biosynthesis and the final pathway enzyme that installs the unusual cercosporin methylenedioxy bridge. Additionally, the expanded cluster contains CFP, which contributes to cercosporin auto-resistance in C. beticola. Together, our results give new insight on the intricate evolution of the CTB cluster.Species in the genus Cercospora cause economically devastating diseases in sugar beet, maize, rice, soy bean and other major food crops. Here we sequenced the genome of the sugar beet pathogen C. beticola and found it encodes 63 putative secondary metabolite gene clusters, including the cercosporin toxin biosynthesis (CTB) cluster. We show that the CTB gene cluster has experienced multiple duplications and horizontal transfers across a spectrum of plant pathogenic fungi, including the wide-host range Colletotrichum genus as well as the rice pathogen Magnaporthe oryzae. Although cercosporin biosynthesis has been thought to-date to rely on an eight gene CTB cluster, our phylogenomic analysis revealed gene collinearity adjacent to the established cluster in all CTB cluster-harboring species. We demonstrate that the CTB cluster is larger than previously recognized and includes cercosporin facilitator protein (CFP) previously shown to be involved with cercosporin auto-resistance, and four additional genes required for cercosporin biosynthesis including the final pathway enzymes that install the unusual cercosporin methylenedioxy bridge. Finally, we demonstrate production of cercosporin by Colletotrichum fioriniae, the first known cercosporin producer within this agriculturally important genus. Thus, our results provide new insight into the intricate evolution and biology of a toxin critical to agriculture and broaden the production of cercosporin to another fungal genus containing many plant pathogens of important crops worldwide.