Yorkie Negatively Regulates the Expression of Antimicrobial Proteins by Inducing Cactus Transcription in Prawns Macrobrachium nipponense.

Ying Huang,Qian Ren,Qin Si,Jie Du

doi:10.3389/fimmu.2022.828271

Abstract

The Hippo signaling pathway controls organ size and immune system in Drosophila and mammals. Yorkie acts as a transcriptional co-activator in the Hippo pathway and cross-talks with other essential pathways. In this study, a Yorkie gene and two Cactus isoforms (designated as MnYorkie, MnCactus-a, and MnCactus-b, respectively) were isolated and characterized from oriental river prawns (Macrobrachium nipponense). Results showed that MnYorkie includes 1620 bp open reading frame and encodes a protein of 539 amino acids (aa). MnCactus-a (377 aa) and MnCactus-b (471 aa) were produced by alternative splicing. MnYorkie and MnCactus were continuously expressed in all selected tissues. Upon Gram-positive bacterium Staphylococcus aureus and Gram-negative bacterium Vibrio parahaemolyticus stimulation, the mRNA levels of MnYorkie and MnCactus in hemocytes and intestines underwent time-dependent enhancement. RNA interference studies showed that MnYorkie silencing remarkably downregulated the transcription of MnCactus but upregulated the expression of seven immune-related genes. In addition, MnYorkie silencing in vivo decreased the susceptibility of prawns to bacterial challenge. After S. aureus and V. parahaemolyticus infection, the survival rate of prawns increased significantly from 2 to 6 days, which corresponded to the period of MnYorkie knockdown. All these findings suggested that MnYorkie in the Hippo pathway might exhibit remarkable biological roles in the immune defense of M. nipponense by negatively regulating the expression of immune-related genes and promoting the transcription of MnCactus.

Highlights

Invertebrates, such as insects and crustaceans, rely mainly on various innate defense reactions to combat pathogen infections [1]
The full-length MnYorkie mRNA (GenBank Accession No OL901207) is 2558 bp long consisting of a 75 bp 5′untranslated region (UTR), an 863 bp 3′-UTR, and a 1620 bp open reading frame (ORF) encoding a predicted polypeptide of 539 amino acids with a calculated Molecular weight (Mw) of 58.3 kDa and a pI of 5.29 (Figure 1A)
Conserved domain analysis revealed that MnYorkie contains a PDB domain 3KYS|D of 110 amino acids located at 16–126 in the N-terminal region, two WW (domain with 2 conserved Trp (W) residues) domains located at 156–188 and 248–280, five low complexity regions located at 127–146, 193–236, 294–320, 378– 404 and 420–437, and a coiled coil region of 24 amino acids located at 341–364 (Figure 1B)

Summary

Introduction

Invertebrates, such as insects and crustaceans, rely mainly on various innate defense reactions to combat pathogen infections [1]. When foreign pathogens invade, the innate immune system triggers various humoral and cellular activities through signal transduction pathways [2, 3]. In canonical Hippo signaling, upstream stimuli activate the Ste-20 family protein kinase Hippo (Hpo), which binds to and phosphorylates regulatory scaffold protein Salvador (Sav) [10]. The Hpo/Sav complex and Mats (Mob as tumor suppressor) adaptor protein phosphorylate and activate Warts (Wts), a nuclear Dbf2-related family protein kinase [11]. Sav, Wts, Mats, and Yki in Drosophila are orthologs of mammalian Ste-20 kinases Mst and Mst (Mst), Sav, large tumor suppressor (Lats) 1/2, Mps one binder 1A and 1B (MOB), and Yesassociated protein (YAP), respectively [14, 15]. Human Mst, Lastl/2, MOB1, and YAP can rescue the corresponding Drosophila mutants and are functionally conserved in mammals [16]

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