Abstract:In plants, perception of invading pathogens involves cell-surface immune receptor kinases. Here, we report that the Arabidopsis SITE-1 PROTEASE (S1P) cleaves endogenous RAPID ALKALINIZATION FACTOR (RALF) propeptides to inhibit plant immunity. This inhibition is mediated by the malectin-like receptor kinase FERONIA (FER), which otherwise facilitates the ligand-induced complex formation of the immune receptor kinases EF-TU RECEPTOR (EFR) and FLAGELLIN-SENSING 2 (FLS2) with their co-receptor BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED KINASE 1 (BAK1) to initiate immune signaling. We show that FER acts as a RALF-regulated scaffold modulating receptor kinase complex assembly. A similar scaffolding mechanism may underlie RALF function in other signalling pathways.One Sentence Summary: A broadly accessible receptor perceives antagonistic endogenous peptides to regulate the formation of plant immune receptor complexes.Main Text: Plant immune pattern recognition receptors (PRRs) are often receptor kinases (1). The Arabidopsis thaliana (hereafter Arabidopsis) receptor kinases FLS2 and EFR bind bacterial flagellin (or the epitope flg22) and EF-Tu (or the epitopes elf18/elf24), respectively, and form ligand-induced complexes with their co-receptor BAK1 (1).

Plant perception of pathogen-associated molecular patterns (PAMPs) triggers a phosphorylation relay leading to PAMP-triggered immunity (PTI). Despite increasing knowledge of PTI signaling, how immune homeostasis is maintained remains largely unknown. Here we describe a forward-genetic screen to identify loci involved in PTI and characterize the Arabidopsis calcium-dependent protein kinase CPK28 as a negative regulator of immune signaling. Genetic analyses demonstrate that CPK28 attenuates PAMP-triggered immune responses and antibacterial immunity. CPK28 interacts with and phosphorylates the plasma-membrane-associated cytoplasmic kinase BIK1, an important convergent substrate of multiple pattern recognition receptor (PRR) complexes. We find that BIK1 is rate limiting in PTI signaling and that it is continuously turned over to maintain cellular homeostasis. We further show that CPK28 contributes to BIK1 turnover. Our results suggest a negative regulatory mechanism that continually buffers immune signaling by controlling the turnover of this key signaling kinase.

During colonization of their hosts, pathogens secrete effector proteins to promote disease development through various mechanisms. Increasing evidence shows that the host microbiome plays a crucial role in health, and that hosts actively shape their microbiomes to suppress disease.We hypothesized that pathogens evolved to manipulate host microbiomes to their advantage in turn. Here, we show that the fungal plant pathogen Verticillium dahliae utilizes effector proteins for niche colonization through selective manipulation of host microbiomes by suppressing microbes with antagonistic activities. Moreover, we show that effector proteins are similarly exploited for microbiome manipulation in the soil environment, where the fungus resides in absence of a host. In conclusion, we demonstrate that pathogens utilize effector proteins to modulate microbiome compositions and propose that their effector catalogs represent an untapped resource for novel antibiotics.

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.

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.Leaf mold disease of tomato (Solanum lycopersicum) is caused by the biotrophic Dothideomycete fungal pathogen Cladosporium fulvum (syn. Passalora fulva and Fulvia fulva) (Thomma et al. 2005). The fungus likely originated in South America, the center of origin for tomato (Jenkins 1948), with Nucleotide sequence data is available in the GenBank database under the following accession numbers: Ecp6, KX943112; Ecp7, KX943113; Ecp8, KX943038; Ecp9-1, KX943041; Ecp9-2, KX943114; Ecp9-3, KX943115; Ecp9-4, KX943116; Ecp9-5, KX943117; Ecp9-6, KX943118; Ecp9-7, KX943119; Ecp9-8, KX943120; Ecp9-9, KX943081; Ecp10-1, KX943046; Ecp10-2, KX943063; Ecp10-3, KX943121; Ecp11-1, KX943050; Ecp12, KX943058; Ecp13, KX943065; Ecp14-1, KX943087; Ecp14-2, KX943122; Ecp15, KX943091; Ecp16, KX943080; Ecp17, KX943051; Ecp18, KX943035; Ecp19-1, KX943036; Ecp19-2, KX943048; Ecp20-1, KX943037; Ecp20-2, KX943057; Ecp20-3, KX943096; Ecp21-1, KX943039; Ecp22, KX943040; Ecp23, KX943044; Ecp24-1, KX943045; Ecp24-2, KX943094; Ecp25, KX943047; Ecp26, KX943052; Ecp27, KX943054; Ecp28-1, KX943056; Ecp28-2, KX943088; Ecp28-3, KX943090; Ecp29, KX943103; Ecp30, KX943076; Ecp31, KX943059; Ecp32-1, KX943062; Ecp32-2, KX943101; Ecp33, KX943066; Ecp34, KX943067; Ecp35, KX943068; Ecp36-1, KX943069; Ecp37, KX943072; Ecp38, KX943073; Ecp39, KX943074; Ecp40, KX943079; Ecp41, KX943082; Ecp42, KX943083; Ecp43-1, KX943089; Ecp44, KX943092; Ecp45, KX943093; Ecp46, KX943095; Ecp47, KX943097; Ecp48, KX943098; Ecp49-1, KX943099; Ecp50-1, KX943100; Ecp51, KX943102; Ecp52, KX943104; Ecp53-1, KX943105; Ecp54-1, KX943107; Ecp55, KX943108; Ecp56, KX943110; Ecp57-1,

Summary In nature, beneficial and pathogenic fungi often simultaneously colonise plants. Despite substantial efforts to understand the composition of natural plant−microbe communities, the mechanisms driving such multipartite interactions remain largely unknown. Here we address how the interaction between the beneficial root endophyte Serendipita vermifera and the pathogen Bipolaris sorokiniana affects fungal behaviour and determines barley host responses using a gnotobiotic soil‐based split‐root system. Fungal confrontation in soil resulted in induction of B. sorokiniana genes involved in secondary metabolism and a significant repression of genes encoding putative effectors. In S. vermifera, genes encoding hydrolytic enzymes were strongly induced. This antagonistic response was not activated during the tripartite interaction in barley roots. Instead, we observed a specific induction of S. vermifera genes involved in detoxification and redox homeostasis. Pathogen infection but not endophyte colonisation resulted in substantial host transcriptional reprogramming and activation of defence. In the presence of S. vermifera, pathogen infection and disease symptoms were significantly reduced despite no marked alterations of the plant transcriptional response. The activation of stress response genes and concomitant repression of putative effector gene expression in B. sorokiniana during confrontation with the endophyte suggest a reduction of the pathogen's virulence potential before host plant infection.

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.

SUMMARYChitin-binding LysM effectors contribute to virulence of various plant pathogenic fungi that are causal agents of foliar diseases. Here, we report on 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 to 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 virulence of V. dahliae strain VdLs17 on tomato, but not on Arabidopsis or on 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 to the core LysM effectors of V. dahliae, this lineage-specific LysM effector of strain VdLs17 contributes to virulence in planta.