# Secreted LysM proteins are required for niche competition and full virulence in Pseudomonas savastanoi during host plant infection

## TL;DR

The study reveals that LysM proteins in Pseudomonas savastanoi are crucial for bacterial movement, division, and competition during olive knot disease infection.

## Contribution

The study identifies novel roles of LysM proteins in bacterial motility, cell division, and niche competition during plant infection.

## Key findings

- LysM1, LysM2, and LysM4 are linked to type IV pilus-related functions and motility.
- LysM3 and LysM5 are essential for cell division and interact with peptidoglycan fragments.
- LysM3 inhibits co-inhabiting Gram-negative bacteria, aiding microbial competition.

## Abstract

Phytopathogenic bacteria secrete diverse virulence factors to manipulate host defenses and establish infection. Characterization of the type III secretion system (T3SS)- and HrpL-independent secretome (T3-IS) in Pseudomonas savastanoi pv. savastanoi (Psv), the causal agent of olive knot disease, identified five secreted LysM-containing proteins (LysM1–LysM5) associated with distinct physiological processes critical for infection. Functional predictions from network analyses suggest that LysM1, LysM2, and LysM4 may participate in type IV pilus-related functions, while LysM3 and LysM5 are likely to possess peptidoglycan hydrolase domains critical for cell division. Supporting these predictions, loss of LysM1 function resulted in impaired twitching and swimming motility, highlighting a role in pilus-mediated movement and early host colonization. In contrast, mutants lacking LysM3 or LysM5 exhibited pronounced filamentation and defective bacterial division, underscoring their essential role in septation, a process crucial for both in planta fitness and tumor formation. Structural modeling and protein stability assays demonstrate that LysM3 interacts with peptidoglycan fragments such as tetra-N-acetylglucosamine and meso-diaminopimelic acid, as well as with zinc ions, through conserved LysM and M23 domains. LysM3 also displayed selective bacteriostatic activity against co-inhabiting Gram-negative bacterial competitors, such as Pantoea agglomerans and Erwinia toletana. Our findings highlight the relevance of LysM proteins in maintaining bacterial integrity, motility, and competitive fitness, which are crucial for successful host infection. This study expands the functional repertoire of LysM-containing proteins and reveals their broader impact on bacterial virulence and adaptation to the plant-associated niche.

Plant pathogenic bacteria secrete a variety of proteins to manipulate host responses and outcompete microbial rivals. In this study, we investigated a group of five secreted proteins with conserved LysM domains produced by the bacterium responsible for olive knot disease, Pseudomonas savastanoi. We found that these proteins fulfill distinct and critical functions during infection. Three are potentially involved in the assembly of type IV pili, structures that help bacteria move and colonize plant tissues, while the other two are related to enzymes that remodel the bacterial cell wall and are essential for proper cell division. Mutants lacking these proteins failed to divide normally and were significantly impaired in their ability to infect olive plants. One of these proteins, LysM3, also exhibited the ability to inhibit other Gram-negative bacteria that coexist with the pathogen in the plant tumor, pointing to a role in microbial competition. These findings shed light on how LysM-containing proteins contribute to both disease progression and bacterial competition and survival in the plant environment. This work paves the way for new insights into bacterial pathogenesis and offers potential strategies for controlling olive knot disease and other plant infections.