The expression of symptoms of viral infections is a byproduct of complex virus-host molecular pathways. These remain largely unknown, especially in the case of fungus-virus pathogen systems. Fungal antiviral responses involve three known mechanisms: RNA interference (RNAi), a post-transcriptional mechanism that inhibits viral replication; transcriptional reprogramming; and recognition of self versus non-self, which limits cell-to-cell transmission of viruses within fungi. While many fungal viruses (mycoviruses) cause asymptomatic infections in their hosts, the mechanisms underlying the induction or suppression of symptoms are not well understood.
Several genetic studies have attempted to explore the fungal factors involved in antiviral responses, but the exact genes and pathways related to symptom induction in fungi remain an open question.
In this context, a research team from Japan, led by Associate Professor Shinji Honda from The Faculty of Medical Sciences, University of Fukui, Japan, and Professor Nobuhiro Suzuki from the Institute of Plant Science and Resources, Okayama University, set out to solve this mystery. They used their recently established fungal virology system in Neurospora crassa to unveil the genes and pathways involved in symptom induction after viral infection. Their findings were made available online on March 24, 2025, and were published in Volume 33, Issue 4 of the prestigious journal Cell Host & Microbe on April 9, 2025.
"In this study, we showed that A-to-I RNA-editing enzymes, whose expression is highly elevated upon viral infection, specifically modify the mRNA of adjacent master transcription factor genes in the fungal genome,” Dr. Honda introduces the main theme of their work. His research team had earlier isolated two viruses that infected N. crassa, for the first time in the world. One of these, Neurospora crassa fusarivirus 1 (NcFV1), is typically asymptomatic in wild-type N. crassa, but causes different symptom profiles in RNAi-deficient mutants, highlighting the complex interplay between virus, host genetics, and symptom development. They later developed this system to conduct mutational and genetic analyses to understand how antiviral responses are controlled in this fungal-viral system.
When the RNAi system is deleted from virus-infected N. crassa, the researchers observed growth defects in the strain compared to wild types, along with exceptionally high levels of viral transcripts in the infected fungi. To understand how switching off the RNAi system triggers such a symptom, they examined the gene expression patterns in this strain. Among the upregulated genes, they specifically noted two genes named old-1 and old-2, which were both revealed to contain deaminase domains.
The team next identified which genomic locations are targeted by the products of old-1 and old-2– OLD-1 and OLD-2. The target locations were approximately 2 kb upstream from old-1/2 in the genome, where it modifies the stop codon of the target transcripts to continue translation and form a full protein with zinc finger domains. These neighboring regions of old-1/2 in the genome were named zao-1/2. Further experiments showed that while OLD-1 is a global RNA editor involved in modifying both zao-1/2 mRNAs, OLD-2 is specific to editing zao-2 mRNA. The regulatory interplay of these genes/proteins in the absence of an RNAi system causes hypersensitive responses in the fungal cells during a viral infection.
The team's investigation into the role of zao-1/2 genes revealed different outcomes depending on which zao genes were present. When infected with the asymptomatic virus NcFV1, wild-type N. crassa, which contains both zao-1 and zao-2 genes, remains asymptomatic, a state associated with specific transcriptional activation of anti-mycovirus responsive genes (AmyREGs). However, the outcome changes in zao gene mutants: in NcFV1-infected mutants lacking only zao-1 (Δzao-1), severe symptoms were observed, possibly due to excessive transcriptional activation, indicating zao-1 is crucial for maintaining the asymptomatic state. More surprisingly, an additional deletion of zao-2 in this background (Δzao-1/2) caused the fungus to recover healthy, becoming asymptomatic. These mutants do not completely induce the AmyREGs that are normally activated during infection. This highlights the critical, but complex, role of zao-1/2 in controlling both symptom development and the activation of the fungal antiviral transcriptional reprogramming.
Why does switching off the RNAi system trigger such a symptom? To unravel this mystery, the researchers investigated the functions of zao-1 and zao-2 in more detail. In asymptomatic wild-type fungi, ZAO-1 is primarily expressed as shorter protein variants (ZAO-1C/1CS) generated by a transcription start site (TSS) switch. These shorter ZAO-1 variants likely compete with full-length ZAO proteins (ZAO-1FL and ZAO-2FL) for DNA binding. This competition is hypothesized to buffer the transcriptional response, maintaining the asymptomatic state. However, in the absence of the RNAi system (in Δqde-2 mutants) or when zao-1 is deleted (in Δzao-1 mutants), the mechanism is altered. In Δqde-2 mutants, OLD enzymes are overexpressed and efficiently edit the premature stop codons in both zao-1 and zao-2 transcripts, leading to increased production of full-length ZAO-1FL and ZAO-2FL. In Δzao-1 mutants, while zao-1 is absent, OLD enzymes efficiently edit the zao-2 transcript, leading to increased ZAO-2FL production. These full-length ZAO proteins, particularly ZAO-2FL, act as potent transcription factors that trigger transcriptional reprogramming, resulting in excessive antiviral responses and symptomatic expression. Furthermore, the absence of ZAO-1 (specifically the short forms ZAO-1C/1CS) eliminates the suppressive competition, allowing the full-length ZAO proteins, particularly ZAO-2FL, to exert stronger transcriptional control, leading to more severe symptomatic responses. Additionally, the team conducted phylogenetic analyses to demonstrate that this RNA editor and its neighboring target transcripts were evolutionarily conserved across several filamentous fungal species, including Neurospora, Fusarium, and Aspergillus.
“This study uncovered a complex layer of antiviral defense involving RNA editing, RNAi, and transcription start site switching, closely linked to transcription reprogramming to regulate symptom induction,” concludes Dr. Honda about the team's work. “Although there are many more questions to be answered in this story of old-zao genomic regulation of antiviral responses in fungi, this study is an important turning point in the development of unique genetic engineering applications and fungal strains with robust antiviral potential.”
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Reference
Title of original paper: RNA editing of genomic neighbors controls antiviral response in fungi
Journal: Cell Host & Microbe
DOI: 10.1016/j.chom.2025.02.016
About University of Fukui, Japan
The University of Fukui is a preeminent research institution with robust undergraduate and graduate schools focusing on education, medical and science, engineering, and global and community studies. The university conducts cutting-edge research and strives to nurture human resources capable of contributing to society on the local, national, and global level.
Website: https://www.u-fukui.ac.jp/eng/
About Associate Professor Shinji Honda from University of Fukui, Japan
Shinji Honda is an Associate Professor at the Faculty of Medical Sciences, University of Fukui, Japan. He received the prestigious Uehara Memorial post-doctoral fellowship to study abroad in 2006, following which he worked as a research associate at the Department of Biology and the Institute of Molecular Biology, University of Oregon, USA. Associate Prof. Honda’s main research interests are in the field of mycoviruses, epigenetics, and Neurospora crassa as a novel fungal system to study antiviral responses. His expertise in the field has resulted in over 35 research papers with more than 1,000 citations.
Funding information
This work was supported by JSPS KAKENHI grant nos. 19H04828, 21H02153, 22K19144, and 22H05593; and 16H06436, 16H06429, 16K21723, and 21H05035; by Research Grants from the University of Fukui; and by the Joint Usage/Research Center, Institute of Plant Science and Resources, Okayama University (nos. R3A1, R419, R519, and R621).
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