Hanna Rovenich

Filamentous pathogen effector functions: of pathogens, hosts and microbiomes

Microorganisms play essential roles in almost every environment on earth. For instance, microbes decompose organic material, or establish symbiotic relationships that range from pathogenic to mutualistic. Symbiotic relationships have been particularly well studied for microbial plant pathogens and have emphasized the role of effectors; secreted molecules that support host colonization. Most effectors characterized thus far play roles in deregulation of host immunity. Arguably, however, pathogens not only deal with immune responses during host colonization, but also encounter other microbes including competitors, (myco)parasites and even potential co-operators. Thus, part of the effector catalog may target microbiome co-inhabitants rather than host physiology.

Functional analysis of the tomato immune receptor Ve1 through domain swaps with its non-functional homolog Ve2.

Resistance in tomato against race 1 strains of the fungal vascular wilt pathogens Verticillium dahliae and V. albo-atrum is mediated by the Ve locus. This locus comprises two closely linked inversely oriented genes, Ve1 and Ve2, which encode cell surface receptors of the extracellular leucine-rich repeat receptor-like protein (eLRR-RLP) type. While Ve1 mediates Verticillium resistance through monitoring the presence of the recently identified V. dahliae Ave1 effector, no functionality for Ve2 has been demonstrated in tomato. Ve1 and Ve2 contain 37 eLRRs and share 84% amino acid identity, facilitating investigation of Ve protein functionality through domain swapping. In this study it is shown that Ve chimeras in which the first thirty eLRRs of Ve1 were replaced by those of Ve2 remain able to induce HR and activate Verticillium resistance, and that deletion of these thirty eLRRs from Ve1 resulted in loss of functionality. Also the region between eLRR30 and eLRR35 is required for Ve1-mediated resistance, and cannot be replaced by the region between eLRR30 and eLRR35 of Ve2. We furthermore show that the cytoplasmic tail of Ve1 is required for functionality, as truncation of this tail results in loss of functionality. Moreover, the C-terminus of Ve2 fails to activate immune signaling as chimeras containing the C-terminus of Ve2 do not provide Verticillium resistance. Furthermore, Ve1 was found to interact through its C-terminus with the eLRR-containing receptor-like kinase (eLRR-RLK) interactor SOBIR1 that was recently identified as an interactor of eLRR-RLP (immune) receptors. Intriguingly, also Ve2 was found to interact with SOBIR1.

Convergent evolution of filamentous microbes towards evasion of glycan‐triggered immunity

896 I. 896 II. 896 III. 897 IV. 898 V. 899 VI. 899 900 References 900 SUMMARY: All filamentous microbes produce and release a wide range of glycans, which are essential determinants of microbe-microbe and microbe-host interactions. Major cell wall constituents, such as chitin and β-glucans, are elicitors of host immune responses. The widespread capacity for glycan perception in plants has driven the evolution of various strategies that help filamentous microbes to evade detection. Common strategies include structural and chemical modifications of cell wall components as well as the secretion of effector proteins that suppress chitin- and β-glucan-triggered immune responses. Thus, the necessity to avoid glycan-triggered immunity represents a driving force in the convergent evolution of filamentous microbes towards its suppression.

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.

Tomato immune receptor Ve1 recognizes surface-exposed co-localized N- and C-termini of Verticillium dahliae effector Ave1

Effectors are secreted by plant pathogens to facilitate infection, often through deregulation of host immune responses. During host colonization, race 1 strains of the soil-borne vascular wilt fungus Verticillium dahliae secrete the effector protein Ave1 that triggers immunity in tomato genotypes that encode the Ve1 immune receptor. Homologs of V. dahliae Ave1 (VdAve1) are found in plants and in few plant pathogenic microbes, and are differentially recognized by Ve1. However, how VdAve1 is recognized by Ve1 remained unknown. Interestingly, C-terminally affinity-tagged versions of VdAve1 failed to activate Ve1-mediated immunity, suggesting that exposure of the C-terminus of VdAve1 is required for Ve1-mediated recognition. This was confirmed by subsequent analysis of C-terminal deletion mutants, and by domain swap experiments. Although required, only the C-terminus of VdAve1 is not sufficient to activate Ve1-mediated immunity. Intriguingly, a three-dimensional structural model of VdAve1 revealed that the N- and C-termini co-localize on a surface-exposed patch of the VdAve1 protein. Indeed, subsequent analyses of N-terminal deletion mutants confirmed that also the N-terminus of VdAve1 is required to activate Ve1-mediated immunity. Thus, we conclude that a surface-exposed patch of the VdAve1 protein that is composed by co-localized N- and C-termini is recognized by the tomato immune receptor Ve1.

Specific Hypersensitive Response–Associated Recognition of New Apoplastic Effectors from Cladosporium fulvum in Wild Tomato

Tomato leaf mold disease is caused by the biotrophic fungus Cladosporium fulvum. During infection, C. fulvum produces extracellular small secreted protein (SSP) effectors that function to promote colonization of the leaf apoplast. Resistance to the disease is governed by Cf immune receptor genes that encode receptor-like proteins (RLPs). These RLPs recognize specific SSP effectors to initiate a hypersensitive response (HR) that renders the pathogen avirulent. C. fulvum strains capable of overcoming one or more of all cloned Cf genes have now emerged. To combat these strains, new Cf genes are required. An effectoromics approach was employed to identify wild tomato accessions carrying new Cf genes. Proteomics and transcriptome sequencing were first used to identify 70 apoplastic in planta-induced C. fulvum SSPs. Based on sequence homology, 61 of these SSPs were novel or lacked known functional domains. Seven, however, had predicted structural homology to antimicrobial proteins, suggesting a possible role in mediating antagonistic microbe-microbe interactions in planta. Wild tomato accessions were then screened for HR-associated recognition of 41 SSPs, using the Potato virus X-based transient expression system. Nine SSPs were recognized by one or more accessions, suggesting that these plants carry new Cf genes available for incorporation into cultivated tomato.

Long Read Annotation (LoReAn): automated eukaryotic genome annotation based on long-read cDNA sequencing

Single-molecule full-length cDNA sequencing can aid genome annotation by revealing transcript structure and alternative splice-forms, yet current annotation pipelines do not incorporate such information. Here we present LoReAn (Long Read Annotation) software, an automated annotation pipeline utilizing short- and long-read cDNA sequencing, protein evidence, and ab initio prediction to generate accurate genome annotations. Based on annotations of two fungal and two plant genomes, we show that LoReAn outperforms popular annotation pipelines by integrating single-molecule cDNA sequencing data generated from either the PacBio or MinION sequencing platforms, and correctly predicting gene structure and capturing genes missed by other annotation pipelines.