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Citrus miraculin-like protein hijacks a viral movement-related p33 protein and induces cellular oxidative stress in defence against Citrus tristeza virus.

ABSTRACT: To defend against pathogens, plants have developed a complex immune system, which recognizes the pathogen effectors and mounts defence responses. In this study, the p33 protein of Citrus tristeza virus (CTV), a viral membrane-associated effector, was used as a molecular bait to explore virus interactions with host immunity. We discovered that Citrus macrophylla miraculin-like protein 2 (CmMLP2), a member of the soybean Kunitz-type trypsin inhibitor family, targets the viral p33 protein. The expression of CmMLP2 was up-regulated by p33 in the citrus phloem-associated cells. Knock-down of the MLP2 expression in citrus plants resulted in a higher virus accumulation, while the overexpression of CmMLP2 reduced the infectivity of CTV in the plant hosts. Further investigation revealed that, on the one hand, binding of CmMLP2 interrupts the cellular distribution of p33 whose proper function is necessary for the effective virus movement throughout the host. On the other hand, the ability of CmMLP2 to reorganize the endomembrane system, amalgamating the endoplasmic reticulum and the Golgi apparatus, induces cellular stress and accumulation of the reactive oxygen species, which inhibits the replication of CTV. Altogether, our data suggest that CmMLP2 employs a two-way strategy in defence against CTV infection.

SUBMITTER: Sun YD

PROVIDER: S-EPMC8131049 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Transient gene expression is a powerful tool to study gene function in plants. In citrus, Agrobacterium transformation is the method of choice for transient expression studies, but this method does not work efficiently with many gene constructs, and there is a need for a more robust transient expression system in citrus leaves. Biolistic particle delivery is an alternative to Agrobacterium transformation, and in some plants, such as Arabidopsis, gives higher transformation rates in leaf tissues than Agrobacterium.Here we describe an improved method for gene expression in epidermal cells of citrus leaves, using the Bio-Rad Helios gene-gun. Gene-gun bombardment of GFP-HDEL produced highly efficient gene expression in large number of cells and in different citrus varieties. We show here that transiently expressed proteins have maintained their functions in plants, and this is demonstrated by the subcellular localization of different organelle markers, and by a functional assay of Xanthomonas citri effector AvrGF1. To further expand the available tools for subcellular localization studies in citrus, we also generated a new set of transgenic citrus plants that contain organelle markers labelling the nuclei, actin and endoplasmic reticulum. Using these new tools, we were able to show that the coat protein of citrus tristeza virus localizes to the cytoplasm and nuclei when expressed in epidermal cells fused to GFP.We have optimized a new method for transient expression in citrus leaves, to give highly reproducible and efficient transformation without producing a high level of injury or artifacts to the bombarded tissue. We also generated the first set organelle markers for use in citrus. These fluorescent protein markers label the nucleus and the actin. With these new resources, protein activity and subcellular localization can be studied in citrus rapidly and in high throughput. The handheld gene-gun device can also be used in the grove to deliver therapies for citrus diseases, such as canker and Huanglongbing, into trees.

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Citrus miraculin-like protein hijacks a viral movement-related p33 protein and induces cellular oxidative stress in defence against Citrus tristeza virus.

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