# Characterization of the Effector Candidate Repertoire in the Arbuscular Mycorrhizal Fungus Rhizophagus irregularis

## TL;DR

This study identifies and characterizes effector proteins in the arbuscular mycorrhizal fungus Rhizophagus irregularis, revealing their potential roles in plant-fungus symbiosis and immune evasion.

## Contribution

Systematic identification and characterization of 227 effector candidates in Rhizophagus irregularis using machine learning and functional assays.

## Key findings

- Most R. irregularis effectors are predicted to localize in the cytoplasm, suggesting roles in symbiotic development.
- A chitin deacetylase effector, RiPDA1, was found to localize in the apoplast and bind chitin, potentially aiding symbiosis.
- Effectors from different microbial species show high species specificity, with distinct clustering patterns.

## Abstract

The majority of terrestrial plants can interact with arbuscular mycorrhizal fungi (AMF) to form symbiotic relationships. AMF colonization not only enhances the host plant’s uptake of mineral nutrients but also improves its tolerance to biotic and abiotic stresses. In return, the host plant supplies the AMF with carbon sources essential for completing its life cycle. How AMF overcome the plant immune system to successfully establish symbiosis has remained an unresolved question. During colonization, AMF also secrete effector proteins, similar to how pathogenic fungi utilize effectors to promote virulence. In this study, we employed machine learning models such as SignalP 6.0 and EffectorP 3.0 to predict potential effectors in Rhizophagus irregularis, leading to the identification of 227 effector candidates. Using EffectorP 3.0, ApoplastP, and LOCALIZER, most R. irregularis effectors were predicted to be localized in the cytoplasm rather than the apoplast, suggesting a functional role in regulating symbiotic development. Only 26% of the predicted effectors were annotated by Pfam, indicating that the majority are proteins of unknown function. Effector proteins from 14 microbial species representing five ecological types (Ectomycorrhizae, Ericoid mycorrhizae, Endophyte, Arbuscular mycorrhizae, and Pathogen) clustered distinctly by species, highlighting the high degree of species specificity among effectors. Two R. irregularis effectors containing the RxLR motif were identified. Although these effectors localized to the cytoplasm, they did not exhibit virulence factor activity. Additionally, we characterized a functionally conserved chitin deacetylase effector, RiPDA1, which localized to the apoplastic space. The Y2H assay indicated that RiPDA1 forms homodimers. The in vitro chitin-binding assay showed that RiPDA1 has an affinity for chitin. RiPDA1 may function as a secretory polysaccharide deacetylase that facilitates symbiosis by deacetylating chitin oligomers. In summary, this study systematically identified and characterized effector proteins in R. irregularis. Similar to pathogenic fungi, AMF appear to employ cell wall-modifying enzymes to overcome plant immune defenses.