The Magnaporthe oryzae effector Pwl2 alters HIPP43 localization to suppress host immunity

Authors: WERE, VINCENT - Sainsbury Laboratory; YAN, XIA - Sainsbury Laboratory; FOSTER, ANDREW - Sainsbury Laboratory; GOMEZ DE LA CRUZ, DIANA - Sainsbury Laboratory; PETIT-HOUDENOT, YOHAN - Sainsbury Laboratory; ESEOLA, ALICE - Sainsbury Laboratory; ZDRZALEK, RAFAL - John Innes Center; RYDER, LAUREN - Sainsbury Laboratory; BENTHAM, ADAM - John Innes Center; KOURELIS, JIORGOS - Sainsbury Laboratory; MENKE, FRANK - Sainsbury Laboratory; SKLENAR, JAN - Sainsbury Laboratory; LANGNER, THORSTEN - Sainsbury Laboratory; GENTLE, AMBER - Sainsbury Laboratory; KAIMENYI, DAVIES - Sainsbury Laboratory; SMOKER, MATTHEW - Sainsbury Laboratory; BAUTISTA, MARK - Sainsbury Laboratory; MA, WEIBIN - Sainsbury Laboratory; MACLEAN, DAN - Sainsbury Laboratory; BANFIELD, MARK - John Innes Center; KAMOUN, SOPHIEN - Sainsbury Laboratory; Moscou, Matthew; TALBOT, NICHOLAS - Sainsbury Laboratory

Submitted to: The Plant Cell
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/12/2025
Publication Date: 5/9/2025
Citation: Were, V., Yan, X., Foster, A., Sklenar, J., Langner, T., Gentle, A., Sahu, N., Bentham, A., Zdrzalek, R., Ryder, L., Kaimenyi, D., Gómez De La Cruz, D., Petit-Houdenot, Y., Eseola, A., Smoker, M., Bautista, M., Ma, W., Kourelis, J., Maclean, D., Banfield, M., Kamoun, S., Menke, F., Moscou, M.J., Talbot, N.J. 2025. The Magnaporthe oryzae effector Pwl2 alters HIPP43 localization to suppress host immunity. The Plant Cell. Article koaf116. https://doi.org/10.1093/plcell/koaf116.
DOI: https://doi.org/10.1093/plcell/koaf116

Interpretive Summary: Plant pathogens manipulate the plants they infect using diverse proteins that they secrete into plant cells. These plant pathogen proteins are called effectors and have diverse roles in suppressing plant immunity, enhancing access to plant-derived nutrients, and facilitating the reproduction of the pathogen. Although the well-known effector PWL2 from the rice blast fungus Magnaporthe oryzae was identified in 1995, it has remained unknown how it is involved in manipulating the plant to benefit the pathogen. In this work, we used new genome analysis techniques and found PWL2 and its family members were routinely present with multiple copies. Applying new gene editing techniques (CRISPR/Cas9), we deleted multiple copies of PWL2 and showed that loss of this effector impacted rice blast fungal growth (the effect is on infecting its host rather than growth of the fungus). Lastly, we used a diverse set of methods to show that Pwl2 suppresses early plant immune responses, likely through its interaction with a plant protein involved in redox homeostasis (reactions involving diverse highly reactive hydrogen/oxygen molecules). This work establishes the approach that Pwl2 uses to contribute to rice blast disease and opens opportunities for breeding plants resistant to rice blast and other diseases.

Technical Abstract: The rice blast fungus Magnaporthe oryzae secretes a battery of effector proteins during host infection to suppress plant immunity and facilitate pathogenesis. Among these effectors, the MAX effector Pwl2 was first identified as a host specificity determinant for infection of weeping lovegrass (Eragrostis curvula) and has been widely studied as a cell biological marker of effector secretion by M. oryzae. Despite its extensive study, the biological function of Pwl2 remains unknown. Here we show that the PWL2 gene is ubiquitous in all host-limited forms of M. oryzae and has undergone substantial copy number expansion, making its functional analysis challenging. Using CRISPR/Cas9 -mediated gene editing, however, we deleted three copies of PWL2 to generate a pwl2 null mutant in M. oryzae strain Guy11, resulting in gain-of-virulence towards weeping lovegrass, but also a significant reduction in the severity of blast symptoms on rice. Transgenic rice and barley lines constitutively expressing PWL2 furthermore resulted in suppression of reactive oxygen species generation and increased susceptibility to blast infection. Co-immunoprecipitation coupled with mass-spectrometry of stable transgenic PWL2 lines led to identification of a heavy metal-binding isoprenylated plant protein HIPP43, as a target of Pwl2. Transgenic lines overexpressing HIPP43 also exhibited attenuated defense responses and increased susceptibility to blast infection, providing evidence that Pwl2 stabilises HIPP43 to modulate immunity. Consistent with this, expression of Pwl2 alters the cellular localisation and abundance of HIPP43. Taken together, our results provide evidence that Pwl2 is a virulence factor in M. oryzae that acts as an important modulator of host immunity.