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

## TL;DR

The study shows that cyclic-di-GMP regulates effector secretion in Pseudomonas syringae, linking it to disease symptoms and bacterial virulence.

## Contribution

The discovery that cyclic-di-GMP binding to HrcN regulates effector secretion and disease symptoms in Pseudomonas syringae.

## Key findings

- Mutating the cyclic-di-GMP binding site in HrcN leads to reduced disease symptoms despite normal bacterial proliferation.
- The effector HopAA1–2 is particularly important for symptom development in Pseudomonas syringae infections.
- Cyclic-di-GMP binding to HrcN may represent a novel regulatory mechanism for effector secretion.

## 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 cyclic-di-GMP binding may represent a novel regulatory mechanism for effector secretion during bacterial infections.

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 the bacterial signalling molecule cyclic-di-GMP, although the purpose of this is currently unknown.

In this study, we use a combination of genetics, plant infection assays and biochemistry to investigate the role of cyclic-di-GMP in HrcN function. We show that mutations in the HrcN cyclic-di-GMP binding site can effectively decouple bacterial proliferation from disease symptoms during infection, producing rare asymptomatic plant infections. This phenomenon is due to reduced secretion of a subset of T3I-effectors in bacteria with mutated HrcN proteins. Of these compromised effectors, one in particular; HopAA1–2, is shown to be highly important for symptom development. Cyclic-di-GMP binding to HrcN may therefore represent a newly discovered mechanism for controlling effector secretion during P. syringae plant infections.