An Integrated Omics Approach Uncovers the Novel Effector Required for Full Virulence of on Tomato

Overview

Journal Front Microbiol

Specialty Microbiology

Date 2022 Jul 22

PMID 35865936

Authors

Mansoor Karimi-Jashni

Kazuya Maeda

Farzaneh Yazdanpanah

Pierre J G M de Wit

Yuichiro Iida

Affiliations

Soon will be listed here.

Abstract

The fungus causes the leaf mould in tomatoes. During the colonization of the host, it secretes plenty of effector proteins into the plant apoplast to suppress the plant's immune system. Here, we characterized and functionally analyzed the gene of using combined omics approaches. RNA-sequencing of susceptible tomato plants inoculated with race 0WU showed strongly induced expression of the gene. Strong upregulation of expression of the gene was confirmed by qPCR, and levels were comparable to those of other known effectors of . The gene encodes a small secreted protein of 149 amino acids with a predicted signal peptide of 17 amino acids. Mass spectrometry of apoplastic fluids from infected tomato leaves revealed the presence of several peptides originating from the Ecp20-2 protein, indicating that the protein is secreted and likely functions in the apoplast. In the genome of , is surrounded by various repetitive elements, but no allelic variation was detected in the coding region of among 120 isolates collected in Japan. Δ deletion mutants of strain 0WU of showed decreased virulence, supporting that Ecp20-2 is an effector required for full virulence of the fungus. Virulence assays confirmed a significant reduction of fungal biomass in plants inoculated with Δ mutants compared to those inoculated with wild-type, Δ-complemented mutants, and ectopic transformants. Sequence similarity analysis showed the presence of homologs in the genomes of several Dothideomycete fungi. The Ecp20-2 protein shows the best 3D homology with the PevD1 effector of , which interacts with and inhibits the activity of the pathogenesis-related protein PR5, which is involved in the immunity of several host plants.

Citing Articles

Beyond the genomes of Fulvia fulva (syn. Cladosporium fulvum) and Dothistroma septosporum: New insights into how these fungal pathogens interact with their host plants.

Mesarich C, Barnes I, Bradley E, de la Rosa S, de Wit P, Guo Y Mol Plant Pathol. 2023; 24(5):474-494.

PMID: 36790136 PMC: 10098069. DOI: 10.1111/mpp.13309.

Characterization of two conserved cell death elicitor families from the Dothideomycete fungal pathogens and (syn. ).

Tarallo M, McDougal R, Chen Z, Wang Y, Bradshaw R, Mesarich C Front Microbiol. 2022; 13:964851.

PMID: 36160260 PMC: 9493481. DOI: 10.3389/fmicb.2022.964851.

References

de Wit P . Apoplastic fungal effectors in historic perspective; a personal view. New Phytol. 2016; 212(4):805-813. DOI: 10.1111/nph.14144. View

Snoeijers S, Perez-Garcia A, Goosen T, de Wit P . Promoter analysis of the avirulence gene Avr9 of the fungal tomato pathogen Cladosporium fulvum in the model filamentous fungus Aspergillus nidulans. Curr Genet. 2003; 43(2):96-102. DOI: 10.1007/s00294-003-0374-7. View

van der Hoorn R, Klemencic M . Plant proteases: from molecular mechanisms to functions in development and immunity. J Exp Bot. 2021; 72(9):3337-3339. PMC: 8042755. DOI: 10.1093/jxb/erab129. View

Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S . MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol. 2011; 28(10):2731-9. PMC: 3203626. DOI: 10.1093/molbev/msr121. View

Zhang Y, Gao Y, Liang Y, Dong Y, Yang X, Qiu D . Verticillium dahliae PevD1, an Alt a 1-like protein, targets cotton PR5-like protein and promotes fungal infection. J Exp Bot. 2018; 70(2):613-626. PMC: 6322577. DOI: 10.1093/jxb/ery351. View

Langmead B, Salzberg S . Fast gapped-read alignment with Bowtie 2. Nat Methods. 2012; 9(4):357-9. PMC: 3322381. DOI: 10.1038/nmeth.1923. View

Joosten M . Isolation of apoplastic fluid from leaf tissue by the vacuum infiltration-centrifugation technique. Methods Mol Biol. 2011; 835:603-10. DOI: 10.1007/978-1-61779-501-5_38. View

Kim D, Pertea G, Trapnell C, Pimentel H, Kelley R, Salzberg S . TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions. Genome Biol. 2013; 14(4):R36. PMC: 4053844. DOI: 10.1186/gb-2013-14-4-r36. View

Thomma B, van Esse H, Crous P, de Wit P . Cladosporium fulvum (syn. Passalora fulva), a highly specialized plant pathogen as a model for functional studies on plant pathogenic Mycosphaerellaceae. Mol Plant Pathol. 2010; 6(4):379-93. DOI: 10.1111/j.1364-3703.2005.00292.x. View

10.

Karimi Jashni M, Mehrabi R, Collemare J, Mesarich C, de Wit P . The battle in the apoplast: further insights into the roles of proteases and their inhibitors in plant-pathogen interactions. Front Plant Sci. 2015; 6:584. PMC: 4522555. DOI: 10.3389/fpls.2015.00584. View

11.

Sanchez-Vallet A, Saleem-Batcha R, Kombrink A, Hansen G, Valkenburg D, Thomma B . Fungal effector Ecp6 outcompetes host immune receptor for chitin binding through intrachain LysM dimerization. Elife. 2013; 2:e00790. PMC: 3700227. DOI: 10.7554/eLife.00790. View

12.

Lu J, Boeren S, de Vries S, van Valenberg H, Vervoort J, Hettinga K . Filter-aided sample preparation with dimethyl labeling to identify and quantify milk fat globule membrane proteins. J Proteomics. 2011; 75(1):34-43. DOI: 10.1016/j.jprot.2011.07.031. View

13.

Cao J, Tan X . Comprehensive Analysis of the Chitinase Family Genes in Tomato (). Plants (Basel). 2019; 8(3). PMC: 6473868. DOI: 10.3390/plants8030052. View

14.

Jaswal R, Kiran K, Rajarammohan S, Dubey H, Singh P, Sharma Y . Effector Biology of Biotrophic Plant Fungal Pathogens: Current Advances and Future Prospects. Microbiol Res. 2020; 241:126567. DOI: 10.1016/j.micres.2020.126567. View

15.

Trapnell C, Williams B, Pertea G, Mortazavi A, Kwan G, van Baren M . Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat Biotechnol. 2010; 28(5):511-5. PMC: 3146043. DOI: 10.1038/nbt.1621. View

16.

van den Burg H, Harrison S, Joosten M, Vervoort J, de Wit P . Cladosporium fulvum Avr4 protects fungal cell walls against hydrolysis by plant chitinases accumulating during infection. Mol Plant Microbe Interact. 2006; 19(12):1420-30. DOI: 10.1094/MPMI-19-1420. View

17.

Rooney H, Vant Klooster J, van der Hoorn R, Joosten M, Jones J, de Wit P . Cladosporium Avr2 inhibits tomato Rcr3 protease required for Cf-2-dependent disease resistance. Science. 2005; 308(5729):1783-6. DOI: 10.1126/science.1111404. View

18.

Petersen T, Brunak S, von Heijne G, Nielsen H . SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat Methods. 2011; 8(10):785-6. DOI: 10.1038/nmeth.1701. View

19.

Balasubramanian V, Vashisht D, Cletus J, Sakthivel N . Plant β-1,3-glucanases: their biological functions and transgenic expression against phytopathogenic fungi. Biotechnol Lett. 2012; 34(11):1983-90. DOI: 10.1007/s10529-012-1012-6. View

20.

Namiki F, Matsunaga M, Okuda M, Inoue I, Nishi K, Fujita Y . Mutation of an arginine biosynthesis gene causes reduced pathogenicity in Fusarium oxysporum f. sp. melonis. Mol Plant Microbe Interact. 2001; 14(4):580-4. DOI: 10.1094/MPMI.2001.14.4.580. View