Title: Antifungal symbiotic peptide NCR044 exhibits unique structure and multifaceted mechanisms of action that confer plant protection

Abstract

In the indeterminate nodules of the model legume Medicago truncatula, ~700 nodule-specific cysteine-rich (NCR) peptides with a conserved cysteine-signature are expressed. NCR peptides are highly diverse in sequence and some of these cationic peptides exhibit antimicrobial activity in vitro and in vivo. However, there is a lack of knowledge regarding their structural architecture, antifungal activity, and modes of action against plant fungal pathogens. Here, the three-dimensional NMR structure of a 36-amino acid NCR peptide, NCR044, was solved. Aside from one four-residue a-helix and one three-residue anti-parallel ß-sheet stabilized by two disulfide bonds, the peptide was otherwise disordered. NCR044 exhibited potent fungicidal activity against multiple plant fungal pathogens including Botrytis cinerea and three Fusarium species. It also inhibited germination in quiescent spores of B. cinerea. In germlings, NCR044 breached the fungal plasma membrane and induced reactive oxygen species generation. NCR044 bound to multiple bioactive phosphoinositides in vitro. In vivo, time-lapse confocal and super-resolution microscopy with NCR044 revealed strong fungal cell wall binding, penetration of the cell membrane at discrete foci followed by gradual loss of turgor, accumulation in the cytoplasm, and elevated levels in nucleoli of germlings. Spray-applied NCR044 significantly reduced gray mold disease symptoms caused by the fungal pathogen B.more » cinerea in tomato and tobacco plants and post-harvest products. This study highlights a novel structurally unique fungal cell penetrating NCR peptide which localized to multiple compartments in a fungal pathogen. Antifungal properties of the peptide reported here warrant further validation of its potential as a peptide-based fungistat/fungicide.« less

Authors:

Velivelli, Siva L. S.  ^[1];

• Search DOE PAGES for author "Velivelli, Siva L. S."
• Search DOE PAGES for ORCID "0000-0002-6831-241X"

Czymmek, Kirk J.  ^[1];

• Search DOE PAGES for author "Czymmek, Kirk J."
• Search DOE PAGES for ORCID "0000-0002-5471-7395"

Li, Hui  ^[1];

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• Search DOE PAGES for ORCID "0000-0002-2489-4905"

Shaw, Jared B. ^[2]; Buchko, Garry W.  ^[3];

• Search DOE PAGES for author "Buchko, Garry W."
• Search DOE PAGES for ORCID "0000-0002-3639-1061"

Shah, Dilip M.  ^[1]

• Search DOE PAGES for author "Shah, Dilip M."
• Search DOE PAGES for ORCID "0000-0001-9503-4729"

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+ Show Author Affiliations

1. Donald Danforth Plant Science Center, St Louis, MO (United States)
2. Pacific Northwest National Lab. (PNNL), Richland, WA (United States)
3. Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Washington State Univ., Pullman, WA (United States)

Publication Date:

Mon Jun 22 00:00:00 EDT 2020

Research Org.:

Pacific Northwest National Lab. (PNNL), Richland, WA (United States)

Sponsoring Org.:

USDOE Office of Science (SC), Biological and Environmental Research (BER); National Science Foundation (NSF)

OSTI Identifier:

1635223

Alternate Identifier(s):

OSTI ID: 1650373

Report Number(s):

PNNL-SA-150641
Journal ID: ISSN 0027-8424

Grant/Contract Number:  

AC05-76RL01830; DBI-1337680

Resource Type:

Accepted Manuscript

Journal Name:

Proceedings of the National Academy of Sciences of the United States of America

Additional Journal Information:

Journal Volume: 117; Journal Issue: 27; Journal ID: ISSN 0027-8424

Publisher:

National Academy of Sciences

Country of Publication:

United States

Language:

English

Subject:

59 BASIC BIOLOGICAL SCIENCES; NCR peptides; NMR structure; antifungal activity; gray mold disease; tomato; structure biology, NMR spectroscopy, antifungal peptides, nodule-specific cysteine-rich peptide, endosymbiotic, nitrogen-fixing bacteroids