Specific hypersensitive response-associated recognition of new apoplastic effectors fromCladosporium fulvumin wild tomato

ABSTRACTTomato leaf mould disease is caused by the biotrophic fungusCladosporium fulvum. During infection,C. fulvumproduces extracellular small secreted protein (SSP) effectors that function to promote colonization of the leaf apoplast. Resistance to the disease is governed byCfimmune 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. fulvumstrains capable of overcoming one or more of all clonedCfgenes have now emerged. To combat these strains, newCfgenes are required. An effectoromics approach was employed to identify wild tomato accessions carrying newCfgenes. Proteomics and transcriptome sequencing were first used to identify 70 apoplasticin planta-inducedC. fulvumSSPs. 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 interactionsin planta. Wild tomato accessions were then screened for HR-associated recognition of 41 SSPs using thePotato virus X-based transient expression system. Nine SSPs were recognized by one or more accessions, suggesting that these plants carry newCfgenes available for incorporation into cultivated tomato.

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

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-05-17-0114-fi ◽

2018 ◽

Vol 31 (1) ◽

pp. 145-162 ◽

Cited By ~ 16

Author(s):

Carl H. Mesarich ◽

Bilal Ӧkmen ◽

Hanna Rovenich ◽

Scott A. Griffiths ◽

Changchun Wang ◽

...

Keyword(s):

Hypersensitive Response ◽

Expression System ◽

Potato Virus X ◽

Cladosporium Fulvum ◽

In Planta ◽

Antimicrobial Proteins ◽

Wild Tomato ◽

Tomato Leaf Mold ◽

Microbe Interactions ◽

Leaf Apoplast

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.

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The coat protein of tobamovirus acts as elicitor of both L 2 and L 4 gene-mediated resistance in Capsicum

Journal of General Virology ◽

10.1099/vir.0.80017-0 ◽

2004 ◽

Vol 85 (7) ◽

pp. 2077-2085 ◽

Cited By ~ 36

Author(s):

P. Gilardi ◽

I. García-Luque ◽

M. T. Serra

Keyword(s):

Hypersensitive Response ◽

Host Response ◽

Expression System ◽

Potato Virus X ◽

Mottle Virus ◽

Coat Proteins ◽

Pepper Mild Mottle Virus ◽

Defence Genes ◽

Glucuronidase Reporter ◽

Capsicum Chacoense

In Capsicum, the resistance conferred by the L 2 gene is effective against all of the pepper-infecting tobamoviruses except Pepper mild mottle virus (PMMoV), whereas that conferred by the L 4 gene is effective against them all. These resistances are expressed by a hypersensitive response, manifested through the formation of necrotic local lesions (NLLs) at the primary site of infection. The Capsicum L 2 gene confers resistance to Paprika mild mottle virus (PaMMV), while the L 4 gene is effective against both PaMMV and PMMoV. The PaMMV and PMMoV coat proteins (CPs) were expressed in Capsicum frutescens (L 2 L 2) and Capsicum chacoense (L 4 L 4) plants using the heterologous Potato virus X (PVX)-based expression system. In C. frutescens (L 2 L 2) plants, the chimeric PVX virus containing the PaMMV CP was localized in the inoculated leaves and produced NLLs, whereas the chimeric PVX containing the PMMoV CP infected the plants systemically. Thus, the data indicated that the PaMMV CP is the only tobamovirus factor required for the induction of the host response mediated by the Capsicum L 2 resistance gene. In C. chacoense (L 4 L 4) plants, both chimeric viruses were localized to the inoculated leaves and produced NLLs, indicating that either PaMMV or PMMoV CPs are required to elicit the L 4 gene-mediated host response. In addition, transient expression of PaMMV CP into C. frutescens (L 2 L 2) leaves and PMMoV CP into C. chacoense (L 4 L 4) leaves by biolistic co-bombardment with a β-glucuronidase reporter gene led to the induction of cell death and the expression of host defence genes in both hosts. Thus, the tobamovirus CP is the elicitor of the Capsicum L 2 and L 4 gene-mediated hypersensitive response.

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Assignment of Amino Acid Residues of the AVR9 Peptide of Cladosporium fulvum That Determine Elicitor Activity

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.1997.10.7.821 ◽

1997 ◽

Vol 10 (7) ◽

pp. 821-829 ◽

Cited By ~ 43

Author(s):

Miriam Kooman-Gersmann ◽

Ralph Vogelsang ◽

Erwin C. M. Hoogendijk ◽

Pierre J. G. M. De Wit

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Resistance Gene ◽

Expression System ◽

Potato Virus X ◽

Cladosporium Fulvum ◽

Amino Acid Residues ◽

Tomato Plants ◽

Loop Region ◽

Relationship Of

The AVR9 peptide of Cladosporium fulvum is an elicitor of the hypersensitive response in tomato plants carrying the Cf-9 resistance gene (MM-Cf9). To determine the structure-activity relationship of the AVR9 peptide, amino acids important for AVR9 elicitor activity were identified by independently substituting each amino acid of AVR9 by alanine. In addition, surface-exposed amino acid residues of AVR9 were substituted by other amino acids. Activity of the mutant Avr9 constructs was studied by expressing the constructs in MM-Cf9 tomato plants, using the potato virus X (PVX) expression system and assessing the severity of necrosis induced by each PVX∷Avr9 construct. This allowed direct identification of amino acid residues of AVR9 that are essential for elicitor activity. We identified amino acid substitutions that resulted in AVR9 mutants with higher, similar, or lower elicitor activity compared to the wild-type AVR9 peptide. Some mutants had completely lost elicitor activity. A selection of peptides, representing different categories, was isolated and injected into leaves of MM-Cf9 plants. The necrosis-inducing activity of the isolated peptides correlated well with the necrosis induced by the corresponding PVX∷Avr9 derivatives. Based on the necrosis-inducing activity of the mutant AVR9 peptides and the global structure of AVR9, we assigned sites in AVR9 that are important for its necrosis-inducing activity. We postulate that the “hydrophobic β-loop” region of the AVR9 peptide is crucial for necrosis-inducing activity in tomato plants that carry the Cf-9 resistance gene.

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Novel System for the Simultaneous Analysis ofGeminivirus DNA Replication and Plant Interactions in Nicotianabenthamiana

Journal of Virology ◽

10.1128/jvi.77.24.13315-13322.2003 ◽

2003 ◽

Vol 77 (24) ◽

pp. 13315-13322 ◽

Cited By ~ 20

Author(s):

Yiguo Hong ◽

John Stanley ◽

Rene van Wezel

Keyword(s):

Defense Mechanism ◽

Intracellular Localization ◽

Potato Virus X ◽

African Cassava Mosaic Virus ◽

Origin Of Replication ◽

Amino Acid Residues ◽

Plant Interactions ◽

In Planta ◽

Gfp Fusion Protein ◽

Host Defense Mechanism

ABSTRACT The origin of replication of African cassava mosaic virus (ACMV) and a gene expression vector based on Potato virus X were exploited to devise an in planta system for functional analysis of the geminivirus replication-associated protein (Rep) in transgenic Nicotiana benthamiana line pOri-2. This line contains an integrated copy of a tandem repeat of the ACMV origin of replication flanking nonviral sequences that can be mobilized and replicated by Rep as an episomal replicon. A Rep-GFP fusion protein can also mobilize and amplify the replicon, facilitating Rep detection in planta. The activity of Rep and its mutants, Rep-mediated host response, and the correlation between Rep intracellular localization and biological functions could be effectively assessed by using this in planta system. Our results indicate that modification of amino acid residues R2, R5, R7 and K11 or H56, L57 and H58 prevent Rep function in replication. This defect correlates with possible loss of Rep nuclear localization and inability to trigger the host defense mechanism resembling a hypersensitive response.

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Affinity-tags are removed from Cladosporium fulvum effector proteins expressed in the tomato leaf apoplast

Journal of Experimental Botany ◽

10.1093/jxb/erj044 ◽

2006 ◽

Vol 57 (3) ◽

pp. 599-608 ◽

Cited By ~ 18

Author(s):

H. Peter van Esse ◽

Bart P. H. J. Thomma ◽

John W. van 't Klooster ◽

Pierre J. G. M. de Wit

Keyword(s):

Tomato Leaf ◽

Effector Proteins ◽

Cladosporium Fulvum ◽

Affinity Tags ◽

Leaf Apoplast

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Pepper Mild Mottle Virus Coat Protein Alone Can Elicit the Capsicum spp. L3 Gene-Mediated Resistance

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.1998.11.12.1253 ◽

1998 ◽

Vol 11 (12) ◽

pp. 1253-1257 ◽

Cited By ~ 23

Author(s):

P. Gilardi ◽

I. García-Luque ◽

M. T. Serra

Keyword(s):

Coat Protein ◽

Resistance Gene ◽

Protein Gene ◽

Expression System ◽

Potato Virus X ◽

Mottle Virus ◽

Capsicum Chinense ◽

Pepper Mild Mottle Virus ◽

Capsicum Spp ◽

Virus Coat

The pepper mild mottle virus (PMMoV-S) (an L3 hypersensitive response [HR]-inducer strain) coat protein was expressed in Capsicum chinense (L3L3) plants with the heterologous potato virus X (PVX)-based expression system. The chimeric virus was localized in the inoculated leaves and induced the HR, thus indicating that the tobamoviral sequences that affect induction of the HR conferred by the L3 resistance gene reside in the coat protein gene. Furthermore, transient expression of the PMMoV-S coat protein in C. chinense leaves by biolistic co-bombardment with a plasmid expressing the β-glucuronidase (GUS) gene leads to the induction of cell death and expression of host defense genes. Thus, the coat protein of PMMoV-S is the elicitor of the Capsicum spp. L3 resistance gene-mediated HR.

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An ancient antimicrobial protein co-opted by a fungal plant pathogen for in planta mycobiome manipulation

10.1101/2021.06.10.447847 ◽

2021 ◽

Author(s):

Nick Snelders ◽

Gabriella Petti ◽

Grardy van den Berg ◽

Michael Seidl ◽

Bart Thomma

Keyword(s):

Life Stage ◽

Antibacterial Properties ◽

Plant Evolution ◽

Effector Proteins ◽

Plant Colonization ◽

Antimicrobial Protein ◽

In Planta ◽

Antimicrobial Proteins ◽

Pathogenic Microbes ◽

Plant Microbiome

Microbes typically secrete a plethora of molecules to promote niche colonization. Soil-dwelling microbes are well-known producers of antimicrobials that are exploited to outcompete microbial co-inhabitants. Also plant pathogenic microbes secrete a diversity of molecules into their environment for niche establishment. Upon plant colonization, microbial pathogens secrete so-called effector proteins that promote disease development. While such effectors are typically considered to exclusively act through direct host manipulation, we recently reported that the soil-borne fungal xylem-colonizing vascular wilt pathogen Verticillium dahliae exploits effector proteins with antibacterial properties to promote host colonization through the manipulation of beneficial host microbiota. Since fungal evolution preceded land plant evolution, we now speculate that a subset of the pathogen effectors involved in host microbiota manipulation evolved from ancient antimicrobial proteins of terrestrial fungal ancestors that served in microbial competition prior to the evolution of plant pathogenicity. Here, we show that V. dahliae has co-opted an ancient antimicrobial protein as effector, named VdAMP3, for mycobiome manipulation in planta. We show that VdAMP3 is specifically expressed to ward off fungal niche competitors during resting structure formation in senescing mesophyll tissues. Our findings indicate that effector-mediated microbiome manipulation by plant pathogenic microbes extends beyond bacteria and also concerns eukaryotic members of the plant microbiome. Finally, we demonstrate that fungal pathogens can exploit plant microbiome-manipulating effectors in a life-stage specific manner, and that a subset of these effectors has evolved from ancient antimicrobial proteins of fungal ancestors that likely originally functioned in manipulation of terrestrial biota.

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