Co-infection of Four Novel Mycoviruses from Three Lineages Confers Hypovirulence on Phytopathogenic Fungus Ustilaginoidea virens

Abstract

Rice false smut caused by Ustilaginoidea virens has become one of the most important diseases of rice. Mycoviruses are viruses that can infect fungi with the potential to control fungal diseases. However, little is known about the biocontrol role of hypoviruses in U. virens. In this study, we revealed that the hypovirulence-associated U. virens strain Uv325 was co-infected by four novel mycoviruses from three lineages, designated Ustilaginoidea virens RNA virus 16 (UvRV16), Ustilaginoidea virens botourmiavirus virus 8 (UvBV8), Ustilaginoidea virens botourmiavirus virus 9 (UvBV9), and Ustilaginoidea virens narnavirus virus 13 (UvNV13), respectively. The U. virens strain co-infected by four mycoviruses showed slower growth rates, reduced conidial yield, and attenuated pigmentation. We demonstrated that UvRV16 was not only the major factor responsible for the hypovirulent phenotype in U. vriens, but also able to prevent U. virens to accumulate more mycotoxin, thereby weakening the inhibitory effects on rice seed germination and seedling growth. Additionally, we indicated that UvRV16 can disrupt the antiviral response of U. virens by suppressing the transcriptional expression of multiple genes involved in autophagy and RNA silencing. In conclusion, our study provided new insights into the biological control of rice false smut.

Introduction

Mycoviruses are a class of viruses that infect and replicate in fungi or oomycetes and are widespread in all major taxa of fungi (Ghabrial and Suzuki 2009; Xie and Jiang 2014; Ghabrial et al. 2015). Since the first definitive report of the cultivated button mushroom Agaricus bisporus infected by mycoviruses in 1962 (Hollings 1962), the exploration of mycoviruses has been ongoing for 61 years. According to the latest list of mycoviruses proposed by the International Committee on Taxonomy of Viruses (ICTV), mycoviruses usually have double-stranded (dsRNA) or single-stranded (ssRNA) genomes (Kotta-Loizou and Coutts 2017), however, DNA viruses have also been identified in recent years in Sclerotinia sclerotiorum and Fusarium graminearum (Yu et al. 2010; Li et al. 2020). Unlike most plant, animal and bacterial viruses, the majority of mycoviruses do not usually attack fungal cells and are asymptomatic in vitro (Xie and Jiang 2014; Ghabrial et al. 2015). Notably, some mycoviruses can reduce the pathogenicity of their fungal hosts, known as hypoviruses, which are an important biocontrol resource because of their hypovirulent characteristics. The most successful instance is the application of Cryphonectria hypovirus 1 (CHV1) in Europe to control the chestnut blight caused by Cryphonectria parasitica (Anagnostakis 1982). In recent years, Zhang et al. have discovered that rapeseed stem rot caused by Sclerotinia sclerotiorum can be effectively controlled by spraying Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1 (SsHADV-1)-infected strain DT-8 in the field (Zhang et al. 2020). SsHADV-1 can convert its fungal host S. sclerotiorum from a typical necrotrophic pathogen to a beneficial endophytic fungus. Furthermore, there are some reports showing that mycoviruses can attenuate fungal pathogenicity, reduce sporulation, alter colony morphology and cause poor mycelial growth (Liu et al. 2022). Therefore, exploring the diversity and biological characteristics of mycoviruses can provide new resources and new insights into the biological control of plant fungal diseases caused by phytopathogenic fungi.

Rice false smut (RFS), caused by ascomycetous fungus Ustilaginoidea virens (Cooke) Takahashi (teleomorph: Villosiclava virens), is a devastating disease in rice-growing regions worldwide. With the widespread cultivation of high-yielding varieties and hybrids, excessive use of chemical fertilizers, and marked changes in global and regional climates, RFS has become one of the most devastating cereal diseases in rice-growing areas worldwide (Sun et al. 2020; He et al. 2022; Zhang et al. 2014b). U. virens not only poses serious threats to global food security, but its mycotoxin also poses threats the health of humans and animals (Koiso et al. 1994; Wang et al. 2017). Mycotoxins are important in the pathogenicity of phytopathogenic fungi and usually play an important role in pathogen colonization and biological processes (Hu et al. 2020). In U. virens, mycotoxins can inhibit the growth and development of rice spikes and the accumulation of total sugars, as well as act as pathogenic factors to help U. virens infect rice (Li et al. 2019; Meng et al. 2019; Sun et al. 2020; Fu et al. 2022). U. virens contaminates rice grain by generating two major types of mycotoxins during infection, including the cyclopeptide ustiloxins and the polyketide ustilaginoidins (Guo et al. 2012; Lu et al. 2014, 2015). In Fusarium graminearum, the production of deoxynivalenol (DON) acts on host plant cell walls, chloroplasts and endoplasmic reticulum and plays a key role in wheat spike, pathogenesis and colonization (Wang et al. 2020). The current main measure to control RFS is still environmentally unfriendly fungicide, which not only results in increased antifungal resistance, but also threatens environmental safety and human health. Therefore, the mycoviruses that infect U. virens have attracted our attention for their high efficiency and persistency. To date, only the genome characterization of dsRNA viruses has been reported in U. virens, including Ustilaginoidea virens RNA virus 1-6 (UvRV1-6) (Zhang et al. 2013a; Zhong et al. 2014a, b; Jiang et al. 2015; Zhong et al. 2017), Ustilaginoidea virens partitivirus 1-4 (UvPV1-4) (Zhang et al. 2013b; Zhong et al. 2014c, 2014a; Jiang et al. 2014), Ustilaginoidea virens nonsegmented virus 1-2 (UvNV1-2) (Zhang et al. 2014a, 2018) and Ustilaginoidea virens unassigned RNA virus HNND-1 (UvURV-HNND-1) (Zhu et al. 2015). Furthermore, our laboratory previously identified 593 mycovirus-related contigs in 182 U. virens strains by metatranscriptomic sequencing (He et al. 2022). However, there are no reports of hypoviruses in U. virens, which attracts us to explore efficient and environmentally friendly novel hypoviruses to control RFS.

Viral co-infections are a common phenomenon in animals and plants and are currently being found in fungi. The interactions between co-infecting mycoviruses may cause different degrees of viral disease symptoms in host fungi via synergistic or antagonistic mechanisms (Hillman et al. 2018; Thapa and Roossinck 2019). For instance, the hypovirulent Sclerotinia sclerotiorum strain Ep-1PN was co-infected by Sclerotinia debilitation-associated RNA virus (SsDRV) and Sclerotinia sclerotiorum RNA virus L (SsRV-L), and the SsDRV is major factor responsible for the hypovirulent phenotype (Jiang et al. 2013). Zhang et al. reported the interaction of two RNA viruses, Yado-nushi virus 1 (YnV1) and Yado-kari virus 1 (YkV1), in the phytopathogenic fungus, Rosellinia necatrix (Zhang et al. 2016). Furthermore, a Fusarium poae strain MAFF 240374 was infected by 16 mycoviruses, including dsRNA, + ssRNA, and −ssRNA viruses (Osaki et al. 2016). In mycoviral co-infection, the synergistic mechanism between multiple viruses was revealed. Co-infection of the Lentinula edodes mycovirus HKB with the Lentinula edodes partitivirus 1 (LePV1) can enhance the accumulation of the LePV1 viral RNA-dependent RNA polymerase (RdRP) (Guo et al. 2017). In contrast, antagonistic mechanisms have also been identified in fungi, when Rosellinia necatrix victorivirus 1 (RnVV1) was co-infected with Mycoreovirus 1 (MyRV1) in C. parasitica, MyRV1 induced the up-regulated expression of the key genes for RNA silencing, DCL2 and AGO2, which activated the RNAi response of the fungal host, inhibiting the replication of RnVV1 (Eusebio Cope and Suzuki 2015). In a previous study, we found that co-infection was prevalent in 182 strains of U. virens collected from Hainan Province, China revealed by metatranscriptomic sequencing (He et al. 2022). In particular, U. virens strain 287 was co-infected by nine different mycoviruses and showed the hypovirulent characteristics (He et al. 2022). The study of co-infecting pathogenic fungi has enabled exploration of viral evolution from a different perspective and identified the main factors responsible for the hypovirulent characteristics.

In this study, we found that a hypovirulent U. virens strain Uv325 was co-infected by four novel mycoviruses from three lineages, designated Ustilaginoidea virens RNA virus 16 (UvRV16), Ustilaginoidea virens botourmiavirus virus 8 (UvBV8), Ustilaginoidea virens botourmiavirus virus 9 (UvBV9), and Ustilaginoidea virens narnavirus virus 13 (UvNV13), respectively. Moreover, we have established an efficient and stable system of viral horizontal transmission to obtain isogenic strains infected by different mycoviruses. Importantly, we showed that UvRV16 is the major factor responsible for the hypovirulent characteristics of U. virens strain Uv325, resulting in slowed growth rates, reduced conidial yield and attenuated pigmentation by suppressing the transcription expression of multiple genes involved in growth, conidiation and pathogenicity. Furthermore, UvRV16 also alleviated the toxic effects of U. virens mycotoxins on rice seed germination and seedling growth. We elucidated a mechanism that UvRV16 RdRP can interact with UvATG6 and suppress the transcriptional expression of autophagy and RNA silencing-related genes to disrupt the antiviral response of U. virens. Therefore, we conclude that UvRV16 is not only a great material for studying the interactions between mycoviruses and host fungi, but also a potential biocontrol resource for control of RFS.

Results

U. virens Strain Uv325 is Coinfected by Four Potentially Novel Mycoviruses

In our previous research (He et al. 2022), a particular Ustilaginoidea virens strain Uv325 (Uv325) attracted our attention by its hypovirulent characteristics. Compared to virulent strain HWD2, strain Uv325 showed abnormal colony morphology, significantly slower growth, reduced conidial production and attenuated pigmentation on potato sucrose agar (PSA) (Fig. 1a-c). Therefore, we speculate that these abnormal phenotypes may be caused by mycoviral infections. We designed specific primers for reverse-transcription PCR (RT-PCR) for the detection of potential mycoviruses in strain Uv325 based on previous metatranscriptomic sequencing data (Table S1) (He et al. 2022). The results demonstrated that strain Uv325 is co-infected by at least four potentially novel mycoviruses which were not detected in virulent strain HWD2 (Fig. 1d).

Fig. 1

Biological characteristics of the U. virens strains Uv325 and HWD2. a Abnormal colony morphology of virus-infected strain Uv325 and virulent strain HWD2 on PSA plates after 14 days of growth in the dark at 28 °C. b Comparison of colony diameters of strains Uv325 and HWD2 on PSA plates after 14 days of growth in the dark at 28 °C. c Comparison of the conidial production ability of strains Uv325 and HWD2 after 7 days of shaking culture in the dark at 28 °C in PS liquid medium. d RT-PCR of total RNAs with mycovirus-specific primers based on metatranscriptomic data. e RT-PCR of dsRNA with mycovirus-specific primers before and after deoxyribonuclease I (DNase I) (digestion of ssDNA and dsDNA) and S1 nuclease (digestion of ssDNA or ssRNA) treatments. Lane M: DL2000 DNA molecular weight marker. The error line indicates the calibration of the mean of the three samples. Different letters at the top of each bar indicate significant differences at P < 0.05 confidence level according to t-test

To confirm the genomic characterization of these four potentially novel mycoviruses, we extracted the viral nucleic acids of strain Uv325 by CF-11 cellulose and treated with different nucleases for RT-PCR. After treatment with deoxyribonuclease I (DNase I) (digestion of ssDNA and dsDNA) and S1 nuclease (digestion of ssDNA or ssRNA), RT-PCR was still able to detect one virus, named Ustilaginoidea virens RNA virus 16 (UvRV16), and the other three undetectable viruses were named Ustilaginoidea virens narnavirus virus 13 (UvNV13), Ustilaginoidea virens botourmiavirus virus 8 (UvBV8) and Ustilaginoidea virens botourmiavirus virus 9 (UvBV9), respectively (Fig. 1e). This indicates that UvRV16 viral nucleic acids are dsRNA molecules, while UvNV13, UvBV8 and UvBV9 viral nucleic acids are + ssRNA molecules. Taken together, these results suggest that the hypovirulent phenotype of U. virens strain Uv325 may be due to the novel mycoviruses co-infection.

Genome Organization of Four Novel Mycoviruses in U. virens Strain Uv325

We characterized the genomic organizations of UvRV16, UvNV13, UvBV8 and UvBV9 by combining metatranscriptomic sequencing data and RNA ligase-mediated (RLM)-rapid amplification of cDNA ends (RACE).

The complete genome of UvRV16 is 5164 bp in length containing two open reading frames (ORFs), ORF1 and ORF2. The tetranucleotide sequence (AUGA) observed in the overlap of the two ORFs, which is typical of members of the genus Victorivirus in the family Totiviridae, is the ORF2 start codon (AUG) and the ORF1 termination codon (UGA) (King et al. 2012; da Silva Camargo et al. 2023). The ORF1 (2259 bp) encodes a capsid protein (CP) with 752 amino acids (aa) that share 44.94% identity with the CP of Helminthosporium victoriae virus 190S (HvV190S, accession number O57043.2). The ORF2 (2580 bp) encodes a viral RNA-dependent RNA polymerase (RdRP) with 859 aa, which is 40.88% identical to the RdRP of Helminthosporium victoriae virus 190S (HvV190S, accession number O57044.2). The coding region is respectively flanked by 5’ and 3’ untranslated regions (UTRs) with lengths of 263 and 66 bp, respectively (Fig. 2a).

Fig. 2

Genome organizations of four mycoviruses UvRV16, UvNV13, UvBV8 and UvBV9. a Schematic genome organization of UvRV16. Bolded boxes indicate the ORFs encoded by UvRV16, and UTRs as gray lines. Representative tetranucleotides (AUGA) are annotated in the schematic. Schematic of genome organizations of UvNV13, UvBV8 and UvBV9 genomes are shown in b and d, respectively. Bold boxes indicate the ORFs encoded by mycoviruses, and gray lines indicate UTRs. c The predicted secondary structure of the 5’-UTR (nucleotides 1–271) and 3’-UTR (nucleotides 1795–1837) of UvNV13. e The predicted secondary structures of the 5’-UTR (nucleotides 1–30) and 3’-UTR (nucleotides 2280–2325) of UvBV8. f The predicted secondary structures of the 5’-UTR (nucleotides 1–45) and 3’-UTR (nucleotides 2375–2415) of UvBV9

The full-length cDNA sequence of UvNV13 is 1837 nt containing one large ORF and possessing short terminal inverted repeats (5’-GGGGC and GCCCC-3’), which are similar to the terminal characteristics of Saccharomyces 23S RNA narnavirus (ScNV-23S) and Saccharomyces 20S RNA narnavirus (ScNV-20S) (Mardanov et al. 2020). The ORF (1524 nt) encodes a RdRP with 507 aa that share 28.09% identity with the ScNV-23S (accession number Q07048.2) and share 25.28% identity with the ScNV-20S (accession number P25328.1) (Fig. 2b). The predicted terminal stem-loop secondary structures showed that the 5’ and 3’ UTRs of UvNV13 could be folded into terminal stable stem-loop structures with ∆G values of −27.60 and −16.00 kcal/mol, respectively (Fig. 2c).

The genomic organizations of UvBV8 and UvBV9 are 2325 and 2415 nt, respectively, and both encode only one large ORF. The ORF of UvBV8 is 1851 nt and encodes a polyprotein with 616 aa, whereas the ORF of UvBV9 is 1968 nt and encodes a polyprotein with 655 aa. However, no similarity viral proteins were found by BLASTp alignment. We performed a BLASTn search with the viral complete genome sequence and found that UvBV8 shared 77.16% identity with the genomic sequence of Pecols virus (PV, accession number MW434079.1), while UvBV9 shared 72.56% identity with the putative RdRP of Magoulivirus sp. (ID: MZ679378.1). Therefore, we speculated that both ORFs of UvBV8 and UvBV9 could encode RdRP, respectively (Fig. 2d). In addition, the predicted terminal stem-loop secondary structures showed that the 5’ and 3’ UTRs of UvBV8 could be folded into terminal stable stem-loop structures with ∆G values of −15.50 and −13.20 kcal/mol, respectively (Fig. 2e). The 5’ and 3’ UTRs of UvBV9 could also be folded into terminal stable stem-loop structures with ∆G values of −13.70 and −22.10 kcal/mol, respectively (Fig. 2f).

Sequence Alignment and Phylogenetic Analyses of Four Potentially Novel Mycoviruses

To clarify the evolutionary relationship between UvRV16 and other mycoviruses, we performed amino acid sequence alignment and phylogenetic classification based on its RdRP protein sequence. Phylogenetic tree was constructed by aligning the RdRP amino acid sequences of UvRV16 with five different viral genera (Victorivirus, Leishmaniavirus, Trichomonasvirus, Totivirus and Giardiavirus) and unclassified viruses in the family Totiviridae (Fig. 3a) (King et al. 2012; da Silva Camargo et al. 2023). The results indicated that UvRV16 formed an independent branch in the genus Victorivirus (Fig. 3a).

Fig. 3

Phylogenetic analyses of four potentially novel mycoviruses. a Phylogenetic tree constructed from the RdRP amino acid sequences of UvRV16 and other viruses in different genus of family Totiviridae, the UvRV16 highlighted with black dot. b UvNV13, UvBV8 and UvBV9 and other viruses in different genus of families Narnaviridae, Botourmiaviridae and Leviviridae. The UvNV13, UvBV8 and UvBV9 highlighted in red with special symbols. Constructed by maximum likelihood (ML) using MEGA 11 software, each branch support was estimated based on 1000 bootstrap repetitions, and bootstrap values (%) were labeled on the branches

Multiple alignment based on the RdRP amino acid sequences of UvRV16 and selected viruses revealed that eight conserved motifs specific to the family Tortiviridae were identified from UvRV16, in addition to the generally conserved GDD motifs (Fig. S1a). Together with the results of overlapping tetranucleotide characterization (AUGA) (Fig. 2a) (Bruenn 1993; Campo et al. 2016; Kartali et al. 2019), multiple alignment and phylogenetic tree, we determined that UvRV16 is a novel mycovirus belonging to the genus Victorivirus in the family Totiviridae.

We have selected the RdRP amino acid sequences of UvBV8, UvBV9, UvNV13 and other 95 mycoviruses from eight genera in three families (Leviviridae, Narnaviridae and Botourmiaviridae) for multiple alignment and phylogenetic classification to clarify the evolutionary relationships of the three single-stranded mycoviruses. Phylogenetic analyses showed that UvBV8 clustered with an unclassified virus, Pecols virus, in the family Botourmiaviridae, UvBV9 clustered with the members in the genus Magoulivirus and UvNV13 clustered with ScNV-23S and ScNV-20S in the genus Narnavirus (Fig. 3b). Multiple alignment showed that the RdRP of UvNV13 is predicted to encode eight conserved motifs unique to the members of the family Nanaviridae (Fig. S1b) (Lin et al. 2020; Fu et al. 2023). Similarly, multiple alignment results showed that eight conserved motifs unique to the family Botourmiaviridae were found in the RdRPs of UvBV8 and UvBV9 (Fig. S1c) (Liu et al. 2020). These RdRPs all contain a highly conserved typical motif “GDD” (motif IV), which is commonly found in + ssRNA mycoviruses and located at the catalytic site of the enzyme to play key role in metal binding (Vázquez et al. 2000; Zhou et al. 2023; O’Reilly and Kao 1998; Argos 1988). Taken together, these results suggest that both UvBV8 and UvBV9 are novel mycoviruses belonging to family Botourmiaviridae, and that UvNV13 is a new member of the genus Narnavirus in the family Nanaviridae.

The Novel Totivirus UvRV16 is a Main Hypovirulent Factor in U. virens

To verify the relationship between four novel mycoviruses and hypovirulence in U. virens, we attempted to obtain isogenic strains infected by one or several viruses using dual-cultures. U. virens strain HWD2-28 with hygromycin B resistance was used as a recipient strain for mycoviruses transmission and it was dual-cultured with hypovirulent U. virens strain Uv325, which was infected with four novel mycoviruses. Strains Uv325 and HWD2-28 were dual-cultured in the same PSA plates, and after 14 days, the mycelial plugs from three different locations were transferred to PSA plates supplemented with hygromycin B and cultured three generations for screening (Fig. S2). Fortunately, we obtained four isogenic strains infected with different viruses by dual-cultures, named strain Uv325-H2-1, strain Uv325-H3-3, strain Uv325-H5-3, and strain Uv325-H6-3. The mycovirus-specific RT-PCR results showed that strain Uv325-H5-3 was infected only by UvBV9 (Fig. 4a). In addition, strain Uv325-H6-3 was co-infected by viruses UvBV8 and UvBV9, and strain Uv325-H2-1 was co-infected by viruses UvRV16, UvBV8 and UvBV9 (Fig. 4a). Furthermore, strain Uv325-H3-3 was co-infected by four novel mycoviruses (Fig. 4a). In addition, to investigate potential vertical transmission of these four mycoviruses in U. virens strain Uv325, we isolated individual conidia from mycelium and cultured on PSA. A total of 20 single subisolates were randomly selected and analyzed for the presence of the four mycoviruses. The experiments showed that all subisolates were infected with all four mycoviruses, confirming effective vertical transmission of these viruses (Fig. S3).

Fig. 4

Biological characteristics of U. virens strain HWD2-28 and its four isogenic strains. a Colony morphology of U. virens strain HWD2-28 and its four isogenic strains cultured on PSA plates at 28 °C for 14 days in the dark. RT-PCR of total RNAs with mycovirus-specific primers. b Comparison of colony diameters of U. virens strain HWD2-28 and its four isogenic strains on PSA plates after 14 days of growth in the dark at 28 °C. c Comparison of the conidial yield of U. virens strain HWD2-28 and its four isogenic strains after 7 days of shaking culture in the dark at 28 °C in PS liquid medium. d Relative expression levels of conidiation-related genes as shown by qRT-PCR. Different letters at the top of each column show significant differences at the P < 0.05 confidence level according to the t-test

To explore the biology of the novel mycovirus, we evaluated the colony morphology, growth rate and conidial yield between the four isogenic strains and the control strain HWD2-28. In terms of colony morphology, after 14 days of cultivation in PSA medium, the pigmentation of strains Uv325-H2-1, Uv325-H3-3, and Uv325-H5-3 were significantly attenuated compared to the strain HWD2-28, which is the main characteristic of hypovirulent (Fig. 4a). Comparison of colony diameters after culturing at 14 days, we found that a significant decrease in the growth rates of strains Uv325-H2-1 and Uv325-H3-3 compared to strain HWD2-28. These two isogenic strains were characterized by all being infected with UvRV16 (Fig. 4a-c). The WT strain had formed 1.8 × 10⁶ conidia/mL after 7 days of culture, whereas strain Uv325-H5-3 and strain Uv325-H6-3 had produced 1.6 × 10⁶ conidia/mL and 1.9 × 10⁶ conidia/mL, respectively, under the same conditions, with no significant difference. In contrast, strain Uv325-H2-1 and strain Uv325-H3-3 had formed 0.18 × 10⁶ conidia/mL and 0.47 × 10⁶ conidia/mL, respectively, after UvRV16 infection, which were significantly decreased compared to the WT strain (Fig. 4c). The expression of eight genes involved in conidiation of filamentous fungi were significantly downregulated in UvRV16-infected strains, including C6 zinc fnger transcription factors UvPRO1, Bax inhibitor-1 (BI-1) protein UvBI-1, transcription factor UvCom1, CMGC kinase UvPmk1 and UvCDC2, the ortholog of yeast HOG1 MAP kinase UvHog1, homology with low-affinity iron transporter protein Uvt3277, homology with mitogen-activated protein kinase UvMK1 (Fig. 4d). Interestingly, we found that the co-infection with UvNV13 could weaken in some degree the effect of UvRV16 on the growth rate and conidial production of U. virens by comparing Uv325-H2-1 and Uv325-H3-3. Taken together, UvRV16 infection resulted in slowed growth rate, reduced conidial yield, and attenuated pigmentation in U. virens strain HWD2-28. We thereby confirmed that the novel totivirus, UvRV16, is the major factor responsible for the hypovirulent phenotype of U. virens.

UvRV16 Reduces Inhibitory Effects of U. virens Mycotoxins on Rice Seed Germination and Seedling Growth

Ustilaginoidins are one of the major types of mycotoxins produced by U. virens, some ustilaginoidins involved in the pathogenesis as virulence factors and were able to interfere the physiological functions of the rice cells (Chen et al. 2021; Zhang et al. 2022; Wen et al. 2023). Studies have shown that Ustilaginoidins O, E, F, R2, B, I and U play important roles in inhibiting rice seedling growth (Lu et al. 2015).

To detect the effects of mycoviruses on the production of mycotoxins in U. virens, we extracted the U. virens crude mycotoxins from PS liquid medium cultures of strains HWD2-28, Uv325-H5-3, Uv325-H6-3, Uv325-H2-1, and Uv325-H3-3, respectively. Rice seeds were treated separately with the crude mycotoxins extracted from the cultures of U. virens strains to determine whether mycoviruses influence the inhibitory effects of mycotoxin compounds on rice seed germination. As shown in Fig. 5a-b, the rice shoots treated with the crude mycotoxins extracted from strain Uv325-H2-1 (co-infected of UvRV16, UvBV8 and UvBV9) were significantly longer than those in other treatments. Whereas the rice shoots treated with the crude mycotoxins extracted from strain Uv325-H6-3 (co-infected of UvBV8 and UvBV9) were shorter those treated with the crude mycotoxins extracted from strain Uv325-H2-1 (co-infected of UvRV16, UvBV8 and UvBV9), suggesting that UvRV16 is a major factor in alleviating the inhibitory effects of mycotoxins on rice seed germination. However, the rice shoots treated with the crude mycotoxins extracted from strain Uv325-H3-3 (co-infected by UvRV16, UvBV8, UvBV9 and UvNV13) were also shorter, suggesting that co-infection with UvNV13 may weaken the inhibitory effect of UvRV16 in some degree. The synthesis gene cluster of ustilaginoidins has been demonstrated to be critical for mycotoxin biosynthesis and modifcations (Li et al. 2019), including the polyketide synthase UvPKS1, transcription initiation factor ugsR1, transcription factor ugsR2, hypothetical protein ugsS and ugsZ, FAD binding domain protein ugsO, major facilitator superfamily transporter ugsT and methyltransferase ugsJ. We found that 8 genes involved in the biosynthesis of mycotoxins in U. virens were significantly down-regulated in strain Uv325-H2-1 (co-infected of UvRV16, UvBV8 and UvBV9) compared to strain Uv325-H6-3 (co-infected of UvBV8 and UvBV9) by qRT-PCR (Fig. 5c).

Fig. 5

UvRV16 interfered the expression of toxin-related genes and reduced the inhibitory effects of U. virens mycotoxins on rice seed germination. a-b The inhibitory effect of mycotoxins of U. virens strain HWD2-28 and four isogenic strains on rice seed germination. Rice seeds were soaked in crude mycotoxins for 2 days and root length was determined. Seeds treated with sterile distilled water were used as control (CK). These experiments were repeated three times with 20 seeds each time. Different letters at the top of each column show significant differences at the P < 0.05 confidence level according to the t-test. c The relative expression levels of eight U. virens mycotoxins synthesis-related genes were verification by qRT-PCR analyses

Furthermore, rice seedlings were treated separately with the crude mycotoxins extracted from the cultures of U. virens strains to explore whether mycoviruses influence the inhibitory effects of mycotoxin compounds on rice seedling growth. We measured the lengths of rice seedlings at 2, 4, 6, and 8 days after mycotoxin treatment (Fig. 6b) and calculated the average growth rates of rice seedlings during 8 days after mycotoxin treatment (Fig. 6c) to evaluate the effect of mycoviruses on mycotoxin production. By comparing the growth dynamics and average growth rates of rice seedlings, we found that crude mycotoxins extracted from strain Uv325-H2-1 (co-infected with UvRV16, UvBV8 and UvBV9) significantly inhibited the growth of rice seedlings, whereas crude mycotoxins extracted from strain Uv325-H6-3 (co-infected with UvBV8 and UvBV9) were not significantly inhibited rice seedling growth. (Fig. 6). Interestingly, the inhibitory effects of the crude mycotoxins extracted from strain Uv325-H3-3 (co-infected by UvRV16, UvBV8, UvBV9 and UvNV13) were also not significant (Fig. 6). These results suggest that UvRV16 is the major factor responsible for attenuating the inhibitory effects of mycotoxins extracted from the cultures of U. virens on rice seedling growth, whereas co-infection with UvNV13 of U. virens strains can weaken the effects of UvRV16 on mycotoxin production in some degree. Taken together, these studies suggest that UvRV16 can suppress the expression of mycotoxin biosynthetic genes to alleviate the inhibitory effect of U. virens crude mycotoxins on rice seed germination and seedling growth.

Fig. 6

UvRV16 significantly suppressed the inhibitory effects of U. virens mycotoxins on rice seedling growth. a The morphology of rice seedlings treated with mycotoxins. The roots of equal-height rice seedlings were soaked in 15 mL-tube containing 5 mL crude mycotoxins, and the height of rice seedlings were measured after 8 days. These experiments were repeated three times at 28 °C under 12 h light/12 h dark. Different letters at the top of each column show significant differences at the P < 0.05 confidence level according to the t-test. b The length of rice seedlings at 2, 4, 6 and 8 days after mycotoxin treatment. c The average growth rates of rice seedlings during 8 days after mycotoxin treatment

UvRV16 Significantly Inhibits the Antiviral Response of U. virens

The important role of autophagy and RNA silencing (RNAi) in plant defense response against plant viruses has been demonstrated (Yang et al. 2023; Huang et al. 2023; Liu et al. 2023), yet their roles in the interactions between mycoviruses and fungal hosts are still unknown. To investigate whether UvRV16 infection regulates antiviral response in U. virens, we first examined the expression of autophagy and RNAi-related genes in strain Uv325-H6-3 (infected with UvBV8 and UvBV9) and strain Uv325-H2-1 (infected with UvRV16, UvBV8 and UvBV9) using qRT-PCR. The results showed that three RNAi-related genes (UvDcl1, UvDcl2 and UvAgo1) and seven autophagy-related genes (UvAtg2, UvAtg4, UvAtg6, UvAtg8, UvAtg9, UvAtg14, and UvVps15) were significantly down-regulated, with the exception of one RNAi-related gene (UvAgo2) which was significantly up-regulated (Fig. 7a), suggesting that UvRV16 infection may suppress host antiviral response. To investigate the mechanism underlying the suppression of autophagy in U. virens by UvRV16, we screened the host factors that interact with UvRV16 through Y2H assay. The results of Y2H assays indicated that the positive protein–protein interaction was found between UvRV16 RdRP and UvATG6, whereas no interaction was evident between UvRV16 RdRP with any other autophagy-related proteins (Fig. 7b). Together, these results suggest that UvRV16 may inhibit U. virens autophagy by interacting with UvATG6, resulting to the promotion of the replication and infection of UvRV16 in U. virens.

Fig. 7

UvRV16 significantly inhibited the antiviral response of U. virens. a qRT-PCR analysis of the transcript levels of autophagy and RNAi-related genes in strain Uv325-H6-3 (infected with UvBV8 and UvBV9) and strain Uv325-H2-1 (infected with UvRV16, UvBV8 and UvBV9). b Yeast two-hybrid (Y2H) assays for possible interactions between UvRV16 RdRP and autophagy-related protein 6 in U. virens (UvATG6)

Discussion

Viruses of the family Totiviridae are dsRNA viruses and the genome contains two large, usually overlapping ORFs that encode RdRP and CP, respectively (Shi et al. 2023). Members of the family Nanaviridae are the simplest genomes and encoding only a single RdRP (Hillman and Cai 2013; Espino Vázquez et al. 2020). The family Botourmiaviridae is a newly established taxonomic taxon in recent years that includes viruses with positive-sense RNA genomes infecting plants and fungi (Ayllón et al. 2020). Here, we identified the co-infection of four novel mycoviruses in a hypovirulence-associated U. virens strain Uv325, named Ustilaginoidea virens RNA virus 16 (UvRV16), Ustilaginoidea virens narnavirus 13 (UvNV13), Ustilaginoidea virens botourmiavirus 8 (UvBV8) and Ustilaginoidea virens botourmiavirus 9 (UvBV9), respectively. The complete genome of UvRV16 is 5164 bp in size and contains two ORFs encoding RdRP and CP, respectively. It is a novel mycovirus belonging to the genus Victorivirus in the family Totiviridae. The UvNV13 is a novel mycovirus belonging to the genus Narnavirus in the family Nanaviridae that is 2302 nt in size and contains a large ORF encoding RdRP. The genomic organizations of UvBV8 and UvBV9 are 2325 and 2415 nt, respectively, and both contain a large ORF encoding RdRP, which are new members belonging to the family Botourmiaviridae. These results suggest that U. virens strain Uv325 harbors a complex and diverse mycovirus community. This research expands our knowledge of the diversity and evolution of viruses in the families Totiviridae, Nanaviridae, and Botourmiaviridae.

Co-infection by two or more mycoviruses is very common among all major taxa of fungi. Jiang et al. reported that U. virens strain GX-1 was co-infected by four mycoviruses, including Ustilaginoidea virens RNA virus 1 (UvRV1) belonging to the family Totiviridae, the unclassified Ustilaginoidea virens RNA virus 4 (UvRV4) and two viruses belonging to the family Partitiviridae (Jiang et al. 2014). Mu et al. characterized nine mycoviruses assigned into eight possible families infecting Sclerotinia sclerotiorum strain SX276. Furthermore, they have applied a series of strategies to eliminate one or more viruses in strain SX276 to verify the relationship between mycoviruses and hypovirulence in S. sclerotiorum (Mu et al. 2021). Their results demonstrated that Sclerotinia sclerotiorum endornavirus 3 (SsEV3) is an important factor responsible for hypovirulent phenotypes in S. sclerotiorum (Mu et al. 2021). In the present study, we obtained isogenic strains infected with different viruses by dual culture to explore the main factors responsible for the hypervirulent phenotype. Compared with the control strain HWD2-28, the growth rates and conidial yields of strains Uv325-H2-1 and Uv325-H3-3 infected with UvRV16 were significantly reduced, whereas the phenotypes of strain Uv325-H6-3 infected with both UvBV8 and UvBV9 were not significantly altered. Interestingly, we found that co-infection with UvNV13 could attenuate in some degree the hypovirulence induced by UvRV16. Based on the qRT-PCR results we hypothesized that UvRV16 may have decreased conidial yield by suppressing the expression of conidiation-related genes in U. virens. This is the first report that identification of mycoviruses with hypovirulent characteristics in U. virens. Therefore, we considered that UvRV16 is a good biocontrol factor for the control of RFS.

False smut balls formed during U. virens infection of rice spikes contain multiple types of mycotoxins, including ustiloxins and ustilaginoidins, which are serious threats to human and animal health (Li et al. 2019). Although the role of mycotoxins in U. virens infection is largely unknown, there are biochemical studies strongly confirming that some pathogenic genes are responsible for the biosynthesis pathway of mycotoxins (Li et al. 2019; Sun et al. 2020; Yang et al. 2023). For example, UvPKS1 was proven to be a key enzyme responsible for the biosynthesis of ustilaginoidin protein (Li et al. 2019). UvHOG1 is not only important for mycelial growth and conidial yield, but also is involved in regulating the production of phytotoxic compounds, and the Uvhog1 mutant reduced the inhibitory properties of mycotoxins on rice shoots (Zheng et al. 2016). Wen et al. reported that AgNPs are an effective nanofungicide against RFS, but AgNPs promote the expression of ustilaginoidin biosynthesis genes, leading to an increase of ustilaginoidins production in U. virens (Wen et al. 2023). Mycotoxins may act as a virulence factor during infection in order to adapt the changes in the infection environment (Zhang et al. 2014b, 2022; Li et al. 2019; Wen et al. 2023; Liu et al. 2023). In this study, we found that UvRV16 attenuated the toxic effects of U. virens mycotoxins on rice seed germination and seedling growth. Therefore, we showed that mycoviruses may be the more suitable biocontrol factors for controlling RFS compared to other control strategies.

In the co-evolutionary arms race between plants and viruses, many defense and counter-defense strategies have been successively established (Hafrén et al. 2018; Li et al. 2018; Wang et al. 2022). To combat viral infections, plants have evolved various complex and systemic defense mechanisms to limit viral replication and transmission. For example, host RNA silencing can target degradation of viral nucleic acids to limit replication after virus infection. Emerging evidence has demonstrated that viral infection also activates host autophagy to selectively degrade viral proteins. For plant viruses, Huang et al. propose a conserved mechanism that plant SnRK1 senses rhabdovirus glycoprotein to initiate autophagy and degrade it via ATG6 to limiting the toxicity of viral glycoprotein and restricting the infection of rhabdovirus (Huang et al. 2023). Turnip mosaic virus (TuMV), a + ssRNA virus, infection activates autophagy in plants, and the core component of autophagy, Beclin1 (ATG6), inhibits viral replication (Li et al. 2018). The capsid protein of Coconut mosaic virus (CaMV) was reported to be targeted degraded by Arabidopsis NBR1 and restricted the viral infection (Hafrén et al. 2017). Yang et al. reported that Barley stripe mosaic virus (BSMV) γb protein inhibits autophagy-mediated antiviral defense and promotes viral replication and infection by competitively disrupting host ATG7-ATG8 interactions (Yang et al. 2018). For human viruses, two proteins encoded by Human cytomegalovirus (HCMV), TRS1 and IRS1, which are characterized by inhibiting autophagy during infection (Mouna et al. 2016). For mycoviruses, transcription levels of C. parasitica genes dcl1 and dcl2 were significantly increased following CHV1 infection (Zhang et al. 2008). Moreover, CHV1 infection of the dcl2 null mutant Δdcl2 or the agl2 null mutant Δagl2 resulted in a severely debilitated growth phenotype and significantly increased virus accumulation, indicating that the genes dcl2 and agl2 are involved in the RNAi antiviral response (Segers et al. 2007; Sun et al. 2009). Conversely, to combat the RNAi antiviral response in C. parasitica, CHV1 encodes the viral suppressor of RNA silencing (VSR), p29, which promotes the accumulation of viral nucleic acids (Segers et al. 2006). In this study, we show that UvRV16 infection significantly suppressed the expression of RNAi and autophagy-related genes in U. virens, and the UvRV16 RdRP interacted with UvAtg6, a core component of the class III PtdIns3K complex (Huang et al. 2023). Therefore, we speculate that virus infection activates the U. virens antiviral pathway, resulting in degradation of viral nucleic acids via RNAi and delivery of viral proteins to autophagosomes for degradation.

Materials and Methods

Fungal Strains and Culture Conditions

The U. virens strain Uv325 was originally isolated from the diseased panicle of rice infected with U. virens, which were collected in Hainan province, China. The U. virens strain HWD2 was kindly provided by Professor Junbin Huang from Huazhong Agriculture University (Hubei province, China). The U. virens strain HWD2-28 is hygromycin B resistance and was used for viral transmission assays in this study. For routine cultures, U. virens strain was grown on potato sucrose agar (PSA, potato 200 g/L, sucrose 20 g/L, agar 17 g/L) medium at 28 °C, and liquid cultures were carried out on potato sucrose (PS, potato 200 g/L, sucrose 20 g/L) medium at 28 °C in the dark with 180 r/min. All strains were stored at 4 °C and −20 °C, respectively.

Nucleic Acid Extraction, Molecular Cloning, Assembly and Analysis

Total RNA was extracted from 100 mg of fresh mycelia using the TransZol Up Plus RNA kit (TransGen Biotech, Beijing, China) according to the manufacturer’s instructions, and dsRNA was extracted using CF-11 cellulose (Sigma) as previously described (Herrero et al. 2012). Total RNA and dsRNA were evaluated for integrity and purity using 1% agarose gel electrophoresis and NanoDrop 2000 (Thermo Scientific, Wilmington, Del., USA), respectively. The extracted dsRNA was treated with DNase I and S1 nuclease (TaKaRa, Dalian, China) to confirm its properties. Extracted nucleic acids were reverse transcribed using TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix kit (TransGen Biotech, Beijing, China).

The 5’ and 3’ terminal unknown sequences of mycoviruses were obtained by reference to a previously reported RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE) (Darissa et al. 2010). PCR amplicons were inserted into pCE2 TA/Blunt-Zero vector (Vazyme Biotech, Nanjing, China) and transformed into Escherichia coli, and positive clones were sequenced at least three times. The complete genome of viruses was assembled by using the DNAMAN software with default parameters. We performed sequence similarity searches by the National Center for Biotechnology Information (NCBI) Blast program (http://blast.st-va.ncbi.nlm.nih.gov/Blast.cgi). The secondary structures of the terminal of viral genomes were predicted by RNAfold Webserver (http://rna.tbi.univie.ac.at//cgi-bin/RNAWebSuite/RNAfold.cgi). To analyze open reading frames (ORFs) in mycovirus genomes, we used the NCBI ORF Finder program (https://www.ncbi.nlm.nih.gov/orffinder/). To obtain the conserved motifs, we predicted the conserved motifs using an online website (http://www.genome.jp/tools/motif/). The amino acid sequences of novel mycoviruses and similar viruses were multiple alignments by an online website (https://smart.embl.de/smart/set_mode.cgi?NORMAL=1) and displayed by SnapGene 6.0.2 software (Letunic et al. 2021). The maximum-likelihood (ML) phylogenetic trees were constructed using MEGA X with 1000 bootstraps (Walker et al. 2015), and were adjusted using the ChiPplot visualisation tool (https://www.chiplot.online/#Phylogenetic-Tree) (Xie et al. 2023).

Viral Horizontal Transmission Assay

To obtain isogenic strains with different viruses, we co-cultured the virus-infected strain Uv325 (donor) with the virus-free strain HWD2-28 (recipient) 1 cm apart on the same PSA dish at 28 °C in dark (Fig. S2a). After culturing for 14 days, mycelial agar plugs were taken from the inner, middle and edges of each colony to obtain recipient-derived strains (Fig. S2b). All recipient-derived strains were transferred to fresh PSA plate containing 200 µg/mL of hygromycin B for three generations and analyzed for the infection of viruses via RT-PCR.

Biological Characterization

To explore the biological effects of mycoviruses on U. virens, we assessed the colony morphology, mycelial growth, and conidiation. For colony morphology and mycelial growth, the agar plugs with a diameter of 5 mm were placed in the center of PSA medium for 14 days of dark at 28 °C. Subsequently, photographs were taken to record colony morphology and the colony diameter were measured using cross measurement method. For the conidial yield assays, four 5 mm diameter agar plugs were inoculated into a 250 mL flask containing 100 mL of liquid PS medium. The samples were incubated at 28 °C for 7 days at 180 r/min. Samples were filtered and used to measure conidial concentration. All of the experiments were repeated three times with three replicates each time.

Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR)

To determine the effect of UvRV16 on the expression of genes related to growth, conidiation, toxins and antivirus in U. vriens, qRT-PCR analyses were performed. qRT-PCR reactions were carried out on a CFX96 TouchTM real-time PCR detection system (Bio-Rad, Hercules, CA, USA) using the ChamQ Universal SYBR qPCR Master Mix (Vazyme Biotech, Nanjing, China). Three independent biological replicates were conducted, each in technical triplicates. The expression level of the gene βTtbulin in U. virens was used as an internal control, and the relative expression levels were calculated by the 2^−ΔΔC(t) method.

Toxicity Assays of Crude Mycotoxins of U. virens

First, the culture filtrates were collected from the cultures of U. virens after 14 days of incubation at 28 °C, 180 rpm/min for preparation of crude mycotoxins according to the previously described method (Fu et al. 2022). The 100 mL of culture filtrates were concentrated to dryness by rotary evaporator at 60 hpa and 60 °C. To adequately extract crude mycotoxins from the dry matter, 100 mL of methanol were mixed with the dry matter and shaken at 150 rpm for 2 h followed by centrifugation at 8000 rpm for 10 min to remove insoluble matter. The supernatant was concentrated to dry extract by a rotary evaporator at 60 °C under 120 hpa. Finally, the dry extract was dissolved in 100 mL of ddH₂O to obtain crude mycotoxins and stored at 4 °C.

For the toxin inhibition of rice seed germination assay, rice seeds were incubated on filter paper soaked with 2 mL of crude mycotoxin as described previously, with 20 rice seeds treated at per time (Zhang et al. 2022). For the crude mycotoxin inhibition of rice seedling growth assays, the roots of equal-height rice seedlings were soaked in a 15 mL-tube containing 5 mL crude mycotoxin. The length of rice seed shoots was measured after 2 days of culture and the height of rice seedlings were measured after 8 days, respectively. These experiments were repeated three times at 28 °C under 12 h light/12 h dark.

Statistical Analysis

Statistical analyses were conducted using the GraphPad Prism version 6.0 (GraphPad Software Inc., La Jolla, CA, USA), and one-way analysis of variance (ANOVA) were used to analyze the significant differences between the control and treatment groups. Significant differences occurred when P < 0.05.