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

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          Significance

          Several nodule-specific cysteine-rich (NCR) peptides expressed in a model legume Medicago truncatula exhibit potent antimicrobial activity. However, their structure–activity relationships and mechanisms of action against fungal pathogens of plants are still largely unknown. A small highly cationic peptide NCR044 with potent antifungal activity has been identified. This peptide has a unique highly dynamic structure and exhibits multifaceted mechanisms of action against a fungal pathogen Botrytis cinerea. Exogenous application of this peptide confers resistance to a gray mold disease caused by B. cinerea in tobacco and tomato plants as well as postharvest products. Our work paves the way for future development of NCR peptides as spray-on antifungal agents.

          Abstract

          In the indeterminate nodules of a model legume Medicago truncatula, ∼700 nodule-specific cysteine-rich (NCR) peptides with 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 the 36-amino acid NCR044 peptide was solved. This unique structure was largely disordered and highly dynamic with one four-residue α-helix and one three-residue antiparallel β-sheet stabilized by two disulfide bonds. NCR044 peptide also exhibited potent fungicidal activity against multiple plant fungal pathogens, including Botrytis cinerea and three Fusarium spp. It inhibited germination in quiescent spores of B. cinerea. In germlings, it breached the fungal plasma membrane and induced reactive oxygen species. It bound to multiple bioactive phosphoinositides in vitro. Time-lapse confocal and superresolution microscopy revealed strong fungal cell wall binding, penetration of the cell membrane at discrete foci, followed by gradual loss of turgor, subsequent 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. cinerea in tomato and tobacco plants, and postharvest products. Our work illustrates the antifungal activity of a structurally unique NCR peptide against plant fungal pathogens and paves the way for future development of this class of peptides as a spray-on fungistat/fungicide.

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          Most cited references39

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          An integrated analysis of plant and bacterial gene expression in symbiotic root nodules using laser-capture microdissection coupled to RNA sequencing.

          Rhizobium-induced root nodules are specialized organs for symbiotic nitrogen fixation. Indeterminate-type nodules are formed from an apical meristem and exhibit a spatial zonation which corresponds to successive developmental stages. To get a dynamic and integrated view of plant and bacterial gene expression associated with nodule development, we used a sensitive and comprehensive approach based upon oriented high-depth RNA sequencing coupled to laser microdissection of nodule regions. This study, focused on the association between the model legume Medicago truncatula and its symbiont Sinorhizobium meliloti, led to the production of 942 million sequencing read pairs that were unambiguously mapped on plant and bacterial genomes. Bioinformatic and statistical analyses enabled in-depth comparison, at a whole-genome level, of gene expression in specific nodule zones. Previously characterized symbiotic genes displayed the expected spatial pattern of expression, thus validating the robustness of our approach. We illustrate the use of this resource by examining gene expression associated with three essential elements of nodule development, namely meristem activity, cell differentiation and selected signaling processes related to bacterial Nod factors and redox status. We found that transcription factor genes essential for the control of the root apical meristem were also expressed in the nodule meristem, while the plant mRNAs most enriched in nodules compared with roots were mostly associated with zones comprising both plant and bacterial partners. The data, accessible on a dedicated website, represent a rich resource for microbiologists and plant biologists to address a variety of questions of both fundamental and applied interest. © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.
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            Plant peptides govern terminal differentiation of bacteria in symbiosis.

            Legume plants host nitrogen-fixing endosymbiotic Rhizobium bacteria in root nodules. In Medicago truncatula, the bacteria undergo an irreversible (terminal) differentiation mediated by hitherto unidentified plant factors. We demonstrated that these factors are nodule-specific cysteine-rich (NCR) peptides that are targeted to the bacteria and enter the bacterial membrane and cytosol. Obstruction of NCR transport in the dnf1-1 signal peptidase mutant correlated with the absence of terminal bacterial differentiation. On the contrary, ectopic expression of NCRs in legumes devoid of NCRs or challenge of cultured rhizobia with peptides provoked symptoms of terminal differentiation. Because NCRs resemble antimicrobial peptides, our findings reveal a previously unknown innovation of the host plant, which adopts effectors of the innate immune system for symbiosis to manipulate the cell fate of endosymbiotic bacteria.
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              Benchmarking fold detection by DaliLite v.5

              Protein structure comparison plays a fundamental role in understanding the evolutionary relationships between proteins. Here, we release a new version of the DaliLite standalone software. The novelties are hierarchical search of the structure database organized into sequence based clusters, and remote access to our knowledge base of structural neighbors. The detection of fold, superfamily and family level similarities by DaliLite and state-of-the-art competitors was benchmarked against a manually curated structural classification. Database search strategies were evaluated using Fmax with query-specific thresholds. DaliLite and DeepAlign outperformed TM-score based methods at all levels of the benchmark, and DaliLite outperformed DeepAlign at fold level. Hierarchical and knowledge-based searches got close to the performance of systematic pairwise comparison. The knowledge-based search was four times as efficient as the hierarchical search. The knowledge-based search dynamically adjusts the depth of the search, enabling a trade-off between speed and recall. http://ekhidna2.biocenter.helsinki.fi/dali/README.v5.html. Supplementary data are available at Bioinformatics online.
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                Author and article information

                Journal
                Proc Natl Acad Sci U S A
                Proc. Natl. Acad. Sci. U.S.A
                pnas
                pnas
                PNAS
                Proceedings of the National Academy of Sciences of the United States of America
                National Academy of Sciences
                0027-8424
                1091-6490
                7 July 2020
                22 June 2020
                22 June 2020
                : 117
                : 27
                : 16043-16054
                Affiliations
                [1] aDonald Danforth Plant Science Center , St Louis, MO 63132;
                [2] bAdvanced Bioimaging Laboratory, Donald Danforth Plant Science Center , St Louis, MO 63132;
                [3] cEarth and Biological Sciences Directorate, Pacific Northwest National Laboratory , Richland, WA 99354;
                [4] dSchool of Molecular Biosciences, Washington State University , Pullman, WA 99164
                Author notes
                1To whom correspondence may be addressed. Email: dshah@ 123456danforthcenter.org .

                Edited by Maria J. Harrison, Cornell University, Ithaca, NY, and approved May 26, 2020 (received for review February 26, 2020)

                Author contributions: S.L.S.V., K.J.C., H.L., G.W.B., and D.M.S. designed research; S.L.S.V., H.L., J.B.S., and G.W.B. performed research; S.L.S.V., K.J.C., H.L., J.B.S., G.W.B., and D.M.S. analyzed data; S.L.S.V., K.J.C., H.L., G.W.B., and D.M.S. wrote the paper.

                Author information
                https://orcid.org/0000-0002-6831-241X
                https://orcid.org/0000-0002-5471-7395
                https://orcid.org/0000-0002-2489-4905
                https://orcid.org/0000-0002-3639-1061
                https://orcid.org/0000-0001-9503-4729
                Article
                202003526
                10.1073/pnas.2003526117
                7354933
                32571919
                04109c2f-d312-4c8c-9941-6375eab982e3
                Copyright © 2020 the Author(s). Published by PNAS.

                This open access article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND).

                History
                Page count
                Pages: 12
                Categories
                Biological Sciences
                Plant Biology

                ncr peptides,nmr structure,antifungal activity,gray mold disease,tomato

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