Cyclic-di-GMP controls Type III effector export and symptom development in Pseudomonas syringae infections via the export ATPase HrcN

Sep 29, 2026

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Pseudomonas syringae is a plant pathogen that delivers host-specific type 3 secreted effectors into plant cytoplasm using its type 3 injectisome (T3I), enabling tissue wetting, immune suppression, and bacterial proliferation that drives chlorosis and necrosis. Effector delivery requires tight temporal control, with HrcN—an export ATPase at the T3I base—being essential for infection. The study tests how disrupting a predicted cyclic-di-GMP–HrcN binding site affects infection and effector secretion, revealing decoupling between proliferation and disease symptoms, linked to reduced translocation of HopAA1–2–containing effector subsets.

LanguageEnglish

CategoryResearch & Report

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Pages20

Reading Time50 min

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OPEN ACCESS Citation: Ward D, Little RH, Thompson CMA, Malone JG (2025) Cyclic-di-GMP controls Type III effector export and symptom development in Pseudomonas syringae infections via the export ATPase HrcN. PLoS Pathog 21(12): e1013376. [https://doi.org/10.1371/journal](https://doi.org/10.1371/journal). ppat.1013376 Editor: Sébastien Bontemps-Gallo, Centre National de la Recherche Scientifique, FRANCE Received: July 14, 2025 Accepted: December 17, 2025 Published: December 26, 2025 Peer Review History: PLOS recognizes the benefits of transparency in the peer review process; therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. The editorial history of this article is available here: [https://doi.org/10.1371/journal](https://doi.org/10.1371/journal). ppat.1013376 Copyright: © 2025 Ward et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, RESEARCH ARTICLE Cyclic-di-GMP controls Type III effector export and symptom development in Pseudomonas syringae infections via the export ATPase HrcN Danny Ward1, Richard H. Little1, Catriona M. A. Thompson1, Jacob G. Malone1,2* 1 John Innes Centre, Norwich Research Park, Norwich, United Kingdom, 2 University of East Anglia, Norwich Research Park, Norwich, United Kingdom * [Jacob.malone@jic.ac.uk](Jacob.malone@jic.ac.uk) Abstract Pseudomonas syringae is a destructive bacterial pathogen that infects a wide variety of plants. Following apoplastic entry, P. syringae uses its type 3 injectisome (T3I) to secrete host-specific effectors into the cytoplasm, enabling tissue wetting and immune suppression and leading to bacterial proliferation, chlorosis and necrosis. P. syringae strains encode dozens of highly specialised effectors, whose composition defines strain specificity and host range. Effective plant infection depends on the tight temporal and hierarchical control of effector delivery through the T3I. Effector secretion is driven by HrcN, an ATPase complex that interacts with the base of the T3I and is essential for plant infection. HrcN binds specifically to the bacterial signalling molecule cyclic-di-GMP, although the impact of binding on T3I function and P. syringae virulence is currently unknown. To address this, we examined the influence of mutating the predicted cyclic-di-GMP-hrcN binding site on plant infection and effector secretion. Despite maintaining effective bacterial proliferation in Arabidopsis thaliana leaves, two hrcN mutants showed severely compromised disease symptoms, a phenotype linked to reduced translocation of a specific subset of T3I-effectors, with HopAA1–2 particularly important for symptom development. We propose that cyclicdi-GMP binding may represent a novel regulatory mechanism for effector secretion during bacterial infections. Author summary Pseudomonas syringae is a bacterial plant pathogen that uses a molecular needle; the type 3 injectisome (T3I), to secrete specialised effector proteins into plant cells. Effector secretion through the T3I is vital for successful infection, enabling bacterial proliferation, immune system suppression and plant tissue degradation. HrcN is an ATPase protein that sits at the base of the T3I, and drives effector delivery through it. We previously showed that HrcN binds specifically to PLOS Pathogens | [https://doi.org/10.1371/journal.ppat.1013376](https://doi.org/10.1371/journal.ppat.1013376) December 26, 2025 1 / 20 and reproduction in any medium, provided the original author and source are credited. Data availability statement: All data used in this submission are contained in the main manuscript and supplementary information files. Funding: JGM, CMAT and RHL were funded by John Innes Centre which was funded by Biotechnology and Biological Sciences Research Council (BBSRC, [https://www](https://www). [ukri.org/

FAQs

What role does HrcN play in Pseudomonas syringae infection?

HrcN is an ATPase complex at the base of the type 3 injectisome and drives effector delivery, which is essential for successful plant infection.

How does cyclic-di-GMP binding to HrcN affect disease symptoms?

Mutations disrupting the predicted cyclic-di-GMP–HrcN binding site maintain effective bacterial proliferation but severely compromise disease symptoms.

Which T3I effectors are most important for symptom development in the hrcN mutants?

A reduced translocation of a specific subset of T3I effectors is linked to the phenotype, with HopAA1–2 particularly important for symptom development.

Sep 29, 2026

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