Unraveling the Nuclear Localization Sequence of MoHTR2, the Nuclear Effector of Magnaporthe oryzae.

Plant pathogenic fungi modulate host immunity by secreting nuclear effectors that interact with host nucleic acids and proteins within the host nucleus. Nuclear effectors are widely known to possess a nuclear localization sequence (NLS) that allows them to enter the host nucleus through either the classical importin α-mediated or non-classical pathways. However, the conserved motif in NLS and the mechanism behind successful nuclear trafficking of fungal nuclear effectors remain largely unexplored. MoHTRs, the nuclear effectors of Magnaporthe oryzae, reprogram the transcription of host immunity-associated genes. Recent research has demonstrated that MoHTR1 requires a classical NLS for importin α-mediated entry into the host nucleus and towards the pathogenicity of M. oryzae. However, the NLS of other fungal nuclear effectors, such as MoHTR2, needs further investigation. In this study, we report that MoHTR2 does not interact with rice importin αs or βs. By performing serial truncation and site-directed mutagenesis, we identified 53HH54 as the core NLS motif essential for the nuclear localization of MoHTR2. We also found that the double histidine in MGG_13063, a nuclear effector candidate of M. oryzae, is involved in its nuclear localization. Deletion of the MoHTR2 core NLS reduced the invasive hyphal growth and lesion formation by M. oryzae. These findings enhance our understanding of the molecular mechanisms underlying the nuclear localization of fungal nuclear effectors and their roles in pathogenicity, contributing to a broader understanding of host-pathogen interactions.