functional-characterization-ring-type-e3-ubiquitin-ligases-vitro

Functional Characterization of RING-Type E3 Ubiquitin Ligases In Vitro and In Planta

Ubiquitination, as a posttranslational modification of proteins, plays an important regulatory role in homeostasis of eukaryotic cells. The covalent ...

University of Idaho

Plant pathogens, such as bacteria, fungi, oomycetes and nematodes, rely on wide range of virulent effectors delivered into host cells to suppress plant immunity. Although phytobacterial effectors have been intensively investigated, little is known about the function of effectors of plant-parasitic nematodes, such as Globodera pallida, a cyst nematode responsible for vast losses in the potato and tomato industries. Here, we demonstrate using in vivo and in vitro ubiquitination assays the potato cyst nematode (Globodera pallida) effector RHA1B is an E3 ubiquitin ligase that employs multiple host plant E2 ubiquitin conjugation enzymes to catalyze ubiquitination. RHA1B was able to suppress effector-triggered immunity (ETI), as manifested by suppression of hypersensitive response (HR) mediated by a broad range of nucleotide-binding leucine-rich repeat (NB-LRR) immune receptors, presumably via E3-dependent degradation of the NB-LRR receptors. RHA1B also blocked the flg22-triggered expression of Acre31 and WRKY22, marker genes of pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI), but this did not require the E3 activity of RHA1B. Moreover, transgenic potato overexpressing the RHA1B transgene exhibited enhanced susceptibility to G. pallida. Thus, our data suggest RHA1B facilitates nematode parasitism not only by triggering degradation of NB-LRR immune receptors to block ETI signaling but also by suppressing PTI signaling via an as yet unknown E3-independent mechanism.

The potato cyst nematode effector RHA1B is a ubiquitin ligase and uses two distinct mechanisms to suppress plant immune signaling.

Plant-parasitic nematodes secrete effectors that manipulate plant cell morphology and physiology to achieve host invasion and establish permanent feeding sites. Effectors from the highly expanded SPRYSEC (SPRY domain with a signal peptide for secretion) family in potato cyst nematodes have been implicated in activation and suppression of plant immunity, but the mechanisms underlying these activities remain largely unexplored. To study the host mechanisms used by SPRYSEC effectors, we identified plant targets of GpRbp-1 from the potato cyst nematode Globodera pallida. Here, we show that GpRbp-1 interacts in yeast and in planta with a functional potato homologue of the Homology to E6-AP C-Terminus (HECT)-type ubiquitin E3 ligase UPL3, which is located in the nucleus. Potato lines lacking StUPL3 are not available, but the Arabidopsis mutant upl3-5 displaying a reduced UPL3 expression showed a consistently small but not significant decrease in susceptibility to cyst nematodes. We observed a major impact on the root transcriptome by the lower levels of AtUPL3 in the upl3-5 mutant, but surprisingly only in association with infections by cyst nematodes. To our knowledge, this is the first example that a HECT-type ubiquitin E3 ligase is targeted by a pathogen effector and that a member of this class of proteins specifically regulates gene expression under biotic stress conditions. Together, our data suggest that GpRbp-1 targets a specific component of the plant ubiquitination machinery to manipulate the stress response in host cells.

Yulin Yuan

Department of Plant Sciences

University of Idaho

Functional Characterization of RING-Type E3 Ubiquitin Ligases In Vitro and In Planta

Yulin Yuan

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University of Idaho

Functional Characterization of RING-Type E3 Ubiquitin Ligases In Vitro and In Planta

Department of Plant Sciences