A Novel Mitovirus PsMV2 Facilitates the Virulence of Wheat Stripe Rust Fungus

Wheat stripe rust, caused by the obligate biotrophic fungus Puccinia striiformis f. sp. tritici (Pst), seriously affects wheat production. Here, we report the complete genome sequence and biological characterization of a new mitovirus from P. striiformis strain GS-1, which was designated as “Puccinia striiformis mitovirus 2” (PsMV2). Genome sequence analysis showed that PsMV2 is 2658 nt in length with an AU-rich of 52.3% and comprises a single ORF of 2348 nt encoding an RNA-dependent RNA polymerase (RdRp). Phylogenetic analysis indicated that PsMV2 is a new member of the genus Unuamitovirus within the family Mitoviridae. In addition, PsMV2 multiplied highly during Pst infection and it suppresses programmed cell death (PCD) triggered by Bax. Silencing of PsMV2 in Pst by barley stripe mosaic virus (BSMV)-mediated Host Induced Gene Silencing (HIGS) reduced fungal growth and decreased pathogenicity of Pst. These results indicate PsMV2 promotes host pathogenicity in Pst. Interestingly, PsMV2 was detected among a wide range of field isolates of Pst and may have coevolved with Pst in earlier times. Taken together, our results characterized a novel mitovirus PsMV2 in wheat stripe rust fungus, which promotes the virulence of its fungal host and wide distribution in Pst which may offer new strategies for disease control.


Total RNA Extraction, cDNA Cloning and Sequencing
Total RNA was extracted from Pst strain GS-1 urediospores. The extracted total RNA was further treated with RQ1 RNase-Free DNase (Promega Corp, Madison, WI, USA) to eliminate fungal DNA. Cloning, sequencing, and analyzing of the cDNAs for the dsRNAs by random primer-mediated PCR were conducted as previously described [38][39][40]. The total RNA was also used for reverse transcription using the RevertAid First Strand cDNA Synthesis Kit for qPCR (Thermo, MA, USA). PCR amplification was performed using Taq PCR Master Mix (CWBIO, Beijing, China). Each purified segment was ligated to the pMD18-T vector (Takara, Dalian, China) for Sanger sequencing. The terminal sequences of PsMV2 were cloned by rapid amplification of ligase-mediated rapid amplification of cDNA ends (RLM-RACE). The PCR primers and their sequences mentioned above are listed in Supplementary Table S1. The full-length cDNA sequence was obtained by assembling the partial sequences in different cDNA clones. In both orientations, each base was determined with at least five independent overlapping clones. A genome of length 2658 nt was obtained from urediospores clones derived from Pst strain GS-1 (race CYR31).

Sequence and Phylogenetic Analysis
Open reading frames (ORFs) and conserved domain(s) were determined using ORF Finder program and CD-search in the National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov, accessed on 3 June 2022), and predictions were made by selecting the "standard" genetic code for all viral contigs. Motifs searches were conducted in the PROSITE database (http://www.expasy.ch/, accessed on 12 October 2022). Protein molecular mass was predicted using the Protein Molecular Weight tool (https://www.bioinformatics. org/sms/prot_mw.html, accessed on 12 October 2022). Potential secondary structures at the terminal sequences of the viral genome sequence were predicted using Mfold RNA structure software [41]. DNAMAN 7.0 and ClustalW 2.0 were used for multiple alignments of nucleotide and coding amino acid sequences [42]. The phylogenetic tree was constructed using the Maximum likelihood (ML) method and the Jones-Taylor-Thornton (JTT) model of the Molecular Evolutionary Genetics Analysis (MEGA) software version 11.0 programs with 1000 bootstrap replicates. See Supplementary Table S2 for all virus names and viral protein accession numbers used for phylogenetic analysis.

RT-PCR Analysis
To assay transcript levels of PsMV2, urediospores and leaves of wheat Su11 infected with urediospores of CYR31 (strain GS-1) were harvested at 6,18,24,48,72,96,120,148,196,216 or 256 h post-inoculation (hpi). To verify the host range of the PsMV2, we performed RT-PCR for a range of plant and fungal samples using a specific primer pair (PsMV2-RdRp-F/R). RNA was extracted from all samples using the Quick RNA Isolation Kit (Huayueyang Biotechnology, Beijing, China). For the cDNA synthesis, 3 µg of RNA was used by the RevertAid First Strand cDNA Synthesis Kit (Thermo, MA, USA). The PCR amplifications were carried out with 2 µL of the synthesized cDNA and the Taq PCR Master Mix (CWBIO, Beijing, China). The products of the RT-PCR amplifications were analyzed by electrophoresis in 1.5% agarose gels. qRT-PCR was performed on a CFX Connect Real-Time System (BioRad, Hercules, CA, USA) with a 20 µL reaction mixture containing 10 µL ChamQ SYBR qPCR Master Mix (Vazyme, Nanjing, China), 2 µL diluted cDNA (1:5), 7 µL distilled H 2 O, 0.5 µL forward primer (10 µM) and 0.5 µL reverse primer (10 µM). The primers are listed in Supplementary Table S1. PstEF1 and TaEF1 were chosen as the internal control genes to normalize the RNA level of Pst and wheat leaves, respectively. Raw data were converted into expression data by the 2 −∆∆Ct method. Each sample was analyzed as three biological replicates and each PCR analysis included three technical replicates.

Agrobacterium Tumefaciens Infiltration Assays
To detect the inhibitory activity of PsMV2 on Bax-induced cell death, A. tumefaciensmediated transient expression method was used. The RdRp nucleotide sequence of PsMV2 was amplified using specific primers PsMV2-PVX-RdRp-F/R (Supplementary Table S1). Coding sequences of PsMV2 were subsequently ligated with potato virus X (PVX) vector pGR106 (for transient expression in N. benthamiana) to construct the PsMV2-GFP plasmid, using ClonExpress II One-Step Cloning Kit (Vazyme, Nanjing, China). This plasmid was separately transformed into A. tumefaciens GV3101 (WEIDI, Shanghai, China), which was cultured in Luria-Bertani medium with 20 mg/L rifampicin and 50 mg/L kanamycin. The infiltration for the tobacco method was described previously [43]. GFP served as a negative control, and then PstGSRE1 can suppress proapoptotic protein Bax-induced PCD used as a positive control. Symptoms were monitored and recorded from 5 to 6 days after infiltration. Each experiment was carried out using three independent biological replicates.

Histological Observation of Pst Growth in Wheat Leaves
The wheat leaves inoculated with BSMV were collected at 24, 48, and 120 hpi for histochemical analysis as previously described [45]. Stained leaf segments were fixed and cleared in ethanol/acetic acid (1:1 v/v) for 3 days and then cleared in saturated chloral hydrate as described previously [46]. H 2 O 2 accumulation was stained with diaminobenzidine (DAB) for microscopic observation (Amresco, Solon, OH, USA). The presence of a substomatal vesicle was defined as an established infection unit. The infection sites and lengths of infection hyphae were stained with wheat germ agglutinin (WGA) conjugated to Alexa-488 (Invitrogen, Carlsbad, CA, USA) and observed under blue light excitation (excitation wavelength 450-480 nm, emission wavelength 515 nm). All microscopic examinations were conducted with an Olympus BX-53 microscope (Olympus Corporation, Tokyo, Japan). The H 2 O 2 accumulation, hyphae lengths, and hyphae infection area (indicating the ability of fungal expansion) were calculated by DP-BSW software (Olympus) connected to the microscope. Fifty infection sites from three randomly selected leaf segments were examined for each treatment.

Protein Extraction and Western Blot
For the Western blot analysis, the total protein was extracted with protein extraction kits (Solarbio, Beijing, China) following the manufacturer's instructions. Protein was separated by 10% SDS-PAGE. Gels were blotted onto a PVDF membrane (Merck Millipore, Burlington, MA, USA) using a transfer buffer at 64 V for 2 h. The procedure of Western blot analysis was described previously [47]. The membranes were washed three times with Tris-buffered saline (TBS, 5 min each) and then blocked with 5% nonfat dry milk in TBST (TBS with 0.1% Tween 20) for 1 h at 25 • C with 50 rpm shaking. Then, the membranes were incubated with primary antibody (mouse anti-GFP antibody (1:10,000, (Proteintech, IL, USA)), mouse anti-Bax antibody (1:10,000, Proteintech)) at 4 • C overnight. After washing three for 5 min in TBST, membranes were then incubated with goat anti-mouse IgG (H+L) antibody (Proteintech) as secondary antibody at 1:10,000 dilutions for 2 h, followed by three washes at room temperature. The Protein bands were detected with chemiluminescence horseradish peroxidase substrate (Merck Millipore, Burlington, MA, USA) and photographed following the manufacturer's instructions.

Complete Sequence and Organization of PsMV2 Genome
The full-length cDNA sequence of Puccinia striiformis mitovirus 2 (PsMV2) was 2658 nt in length, with a G+C content of 47.7%, which is similar to the mitochondrial genomes of fungi and plants, and shares characteristics with members of the genus Unuamitovirus. The cDNA sequence of PsMV2 was deposited in GenBank under the accession number OQ263177. It contains a single ORF being 2349 nt long that initiated at the nucleotide position 226 and terminated at position 2574, potentially encoding 783 amino acids (aa) residues with a calculated molecular weight of 88.24 kDa between the initiation AUG triplet and the termination UAA triplet ( Figure 1A). In addition, the genome of PsMV2 had a 5 -untranslated region (UTR) of 225 nt and a 3 -UTR of 84 nt ( Figure 1A). As with the rest of mitoviruses, the genome of PsMV2 presents a high percentage of A+U (52.3%) and contains a number of UGA and UGG codons that encode the amino acid tryptophan rather than acting as stop codons as in the universal genetic code. The ORF contains multiple codons (43 UGA and 47 UGG, UGA/UGG ratio < 0). The stem-loop structures of the 5 and 3 UTRs of PsMV2 were predicted using the Mfold program (http://rna.tbi.univie.ac.at//cgi-bin/RNAWebSuite/ RNAfold.cgi, accessed on 12 October 2022), with initial ∆G values of −11.9 kcal/mol and −10.0 kcal/mol, respectively ( Figure 1B). Additionally, the sequences of the 5 and 3 UTRs are inverted complementary, and have a potentially stable panhandle structure with a G value of −21.0 kcal/mol ( Figure 1B).

Phylogenetic Analysis of the PsMV2
Based on the conserved domain database (CDD) search on the ORF-encoded RNAdependent RNA polymerase (RdRp), the conserved RdRp domain belongs to the family Mitoviridae (RdRp: 180-466 aa). Multiple sequence alignment revealed that the RdRp of PsMV2 contains six conserved motifs (I-VI) (Figure 2A), which is a specific characteristic of the genus Unuamitovirus [48]. A homology search with BLASTP showed that this 88.24 kDa protein was most closely related to the RNA-dependent RNA polymerases (RdRps) of Cronartium ribicola mitovirus 1, Puccinia striiformis mitovirus 1 and Helicobasidium mompa mitovirus 1-18 (Supplementary Table S3). The virus identified in the Pst strain GS-1 (race CYR31) exhibited the highest similarity to Cronartium ribicola mitovirus 1 with 48% identity. The species demarcation criteria in the genus Mitovirus, defined by the ICTV 9th Report (https://talk.ictvonline.org/, accessed on 12 January 2023), indicate that mitoviruses with homologies in the amino acid sequence of RdRp proteins greater than 90% belong to different strains of the same mitovirus species. Therefore, we can conclude that the PsMV2 in the Pst strain GS-1 (race CYR31) corresponds to a novel mycovirus, a tentative member of a new species, belonging to the genus Unuamitovirus of mitochondrial mycoviruses with +ssRNA genomes. We have named this novel mycovirus, the first reported in this fungal pathogen, Puccinia striiformis mitovirus 2 (PsMV2). To establish the relationship between PsMV2 and other mycoviruses, a Maximum likelihood (ML) phylogenetic tree was constructed based on the RdRp aa sequence of PsMV2 and other related viral sequences, including members of the family Mitoviridae and representative members of the family Narnaviridae ( Figure 2B). In the obtained phylogenetic tree, PsMV2 was grouped with unuamitoviruses, such as the Cronartium ribicola mitovirus 1 ( Figure 2B). Therefore, we concluded that PsMV2 was a new member of the recently proposed genus of Unuamitovirus within the family Mitoviridae [16].

Phylogenetic Analysis of the PsMV2
Based on the conserved domain database (CDD) search on the ORF-encoded RNA dependent RNA polymerase (RdRp), the conserved RdRp domain belongs to the family Mitoviridae (RdRp: 180-466 aa). Multiple sequence alignment revealed that the RdRp o PsMV2 contains six conserved motifs (I-VI) (Figure 2A), which is a specific characteristic of the genus Unuamitovirus [48]. A homology search with BLASTP showed that this 88.24 kDa protein was most closely related to the RNA-dependent RNA polymerases (RdRps of Cronartium ribicola mitovirus 1, Puccinia striiformis mitovirus 1 and Helicobasidium mompa mitovirus 1-18 (Supplementary Table S3). The virus identified in the Pst strain GS-1 (race CYR31) exhibited the highest similarity to Cronartium ribicola mitovirus 1 with 48% identity. The species demarcation criteria in the genus Mitovirus, defined by the ICTV 9th Report (https://talk.ictvonline.org/, accessed on 12 January 2023), indicate that mi toviruses with homologies in the amino acid sequence of RdRp proteins greater than 90% belong to different strains of the same mitovirus species. Therefore, we can conclude tha the PsMV2 in the Pst strain GS-1 (race CYR31) corresponds to a novel mycovirus, a tenta tive member of a new species, belonging to the genus Unuamitovirus of mitochondrial my coviruses with +ssRNA genomes. We have named this novel mycovirus, the first reported in this fungal pathogen, Puccinia striiformis mitovirus 2 (PsMV2). To establish the rela tionship between PsMV2 and other mycoviruses, a Maximum likelihood (ML) phyloge netic tree was constructed based on the RdRp aa sequence of PsMV2 and other related viral sequences, including members of the family Mitoviridae and representative member of the family Narnaviridae ( Figure 2B). In the obtained phylogenetic tree, PsMV2 wa grouped with unuamitoviruses, such as the Cronartium ribicola mitovirus 1 ( Figure 2B) Therefore, we concluded that PsMV2 was a new member of the recently proposed genu of Unuamitovirus within the family Mitoviridae [16].

Transcript Level of PsMV2 Is Upregulated during the Early Infection Stage
To investigate the transcript level of PsMV2 during Pst infection stages, quantitative RT-PCR (qRT-PCR) was used. Results indicated that the transcription level of PsMV2 increased approximately 5.4-9 fold compared to urediospores during the penetration stages of Pst strain GS-1 (race CYR31) on wheat, 6-24 hpi ( Figure 3). However, it decreased to about 1.6-5 fold during 48-96 hpi and was barely detectable at 120-256 hpi ( Figure 3). Therefore, it was found that the transcription level of PsMV2 increased during the early infection stages of the host fungus, especially during haustorial and hyphal formation, and decreases drastically during the late "parasitic/biotrophic" stage of infection and sporulation.

PsMV2 Suppressed Bax-Induced Cell Death
Because Pst is not flexible for transformation, we used the transient expression method mediated by A. tumefaciens to verify the ability of PsMV2 to suppress Bax-triggered PCD, which is similar to the defense-related HR in plant cells [49,50]. One criterion for the suppression of plant immunocompetence is the inhibition of programmed cell death [51]. PstGSRE1 which could suppress Bax-triggered PCD in N. benthamiana was used as a positive control [43]. For the negative control (only GFP), they did not abolish the function of Bax ( Figure 4A). When chlorophyll was removed from tobacco leaves, macroscopic cell death was observed in the negative zone ( Figure 4A). By contrast, we observed that PsMV2 could also suppress Bax-triggered PCD ( Figure 4A). The protein expression of PsMV2-GFP, GFP and Bax were detected by Western blot with an anti-GFP antibody/anti-Bax antibody ( Figure 4B), which indicated that PsMV2 was responsible for the suppression of PCD induced by Bax.   RT-PCR (qRT-PCR) was used. Results indicated that the transcription level of PsMV2 increased approximately 5.4-9 fold compared to urediospores during the penetration stages of Pst strain GS-1 (race CYR31) on wheat, 6-24 hpi (Figure 3). However, it decreased to about 1.6-5 fold during 48-96 hpi and was barely detectable at 120-256 hpi ( Figure 3). Therefore, it was found that the transcription level of PsMV2 increased during the early infection stages of the host fungus, especially during haustorial and hyphal formation, and decreases drastically during the late "parasitic/biotrophic" stage of infection and sporulation. Urediospores were used as a control. Relative transcript level of PsMV2 was calculated using the comparative 2 −ΔΔCT method. PstEF1-F/R was used as the reference gene. Mean and standard deviation were calculated with data from three independent biological replicates. Differences were assessed using Dunnett s test, and asterisks indicate p < 0.05.

PsMV2 Suppressed Bax-Induced Cell Death
Because Pst is not flexible for transformation, we used the transient expression method mediated by A. tumefaciens to verify the ability of PsMV2 to suppress Bax-triggered PCD, which is similar to the defense-related HR in plant cells [49,50]. One criterion for the suppression of plant immunocompetence is the inhibition of programmed cell death [51]. PstGSRE1 which could suppress Bax-triggered PCD in N. benthamiana was used as a positive control [43]. For the negative control (only GFP), they did not abolish the function of Bax ( Figure 4A). When chlorophyll was removed from tobacco leaves, macroscopic cell death was observed in the negative zone ( Figure 4A). By contrast, we observed that PsMV2 could also suppress Bax-triggered PCD ( Figure 4A). The protein expression of PsMV2-GFP, GFP and Bax were detected by Western blot with an anti-GFP antibody/anti-Bax antibody ( Figure 4B), which indicated that PsMV2 was responsible for the suppression of PCD induced by Bax. Urediospores were used as a control. Relative transcript level of PsMV2 was calculated using the comparative 2 −∆∆CT method. PstEF1-F/R was used as the reference gene. Mean and standard deviation were calculated with data from three independent biological replicates. Differences were assessed using Dunnett's test, and asterisks indicate p < 0.05.

Silencing of PsMV2 Significantly Reduces the Virulence of Pst
To determine whether PsMV2 is involved in the pathogenicity of Pst infection in wheat, barley stripe mosaic virus (BSMV)-mediated HIGS was used to silence the expression of PsMV2 in Pst during infection. For the BSMV-HIGS assay, two cDNA fragments of the PsMV2 gene were silenced individually, the constructs were designated BSMV: PsMV2-1as and BSMV: PsMV2-2as, respectively ( Figure 5A). At 12 days after BSMV inoculation, all of the Su11 wheat infected with BSMV: γ, BSMV: PsMV2-1/2as expressed sim-

Silencing of PsMV2 Significantly Reduces the Virulence of Pst
To determine whether PsMV2 is involved in the pathogenicity of Pst infection in wheat, barley stripe mosaic virus (BSMV)-mediated HIGS was used to silence the expression of PsMV2 in Pst during infection. For the BSMV-HIGS assay, two cDNA fragments of the PsMV2 gene were silenced individually, the constructs were designated BSMV: PsMV2-1as and BSMV: PsMV2-2as, respectively ( Figure 5A). At 12 days after BSMV inoculation, all of the Su11 wheat infected with BSMV: γ, BSMV: PsMV2-1/2as expressed similar phenotypes of mild chlorotic mosaic symptoms on the fourth leaf. Su11 seedlings inoculated with BSMV: TaPDS exhibited strong photobleaching symptoms, confirming that the BSMV-mediated gene silencing system functioned correctly and could be used in further experiments ( Figure 5B). Subsequently, the fourth leaf of wheat was inoculated with fresh ungerminated urediospores of CYR31 (GS-1), and rust phenotypes were photographed at 14 dpi. After Pst inoculation, the number of rust pustules was significantly reduced in the wheat leaves inoculated with BSMV: PsMV2-1/2as compared with the control plants ( Figure 5C). Total genomic DNA was isolated from wheat leaves infected with Pst, and the relative levels of PsEF1 and TaEF1 were quantified by qRT-PCR. At 10 days after inoculation, relative to the control plants, the biomass ratio of Pst in leaves treated with BSMV: PsMV2-1as and BSMV: PsMV2-2as were significantly reduced by 36% and 57%, respectively ( Figure 5E). To test the silencing efficiency of BSMV-HIGS, qRT-PCR was used to assay the relative transcript level of PsMV2 at 24, 48, and 120 hpi with Pst race CYR31 (strain GS-1). Compared with control plants, the transcript level of BSMV: PsMV2-1as-infected leaves were reduced by 83%, 80% and 45% at 24, 48, and 120 hpi, respectively ( Figure 5D). Similarly, in BSMV: PsMV2-2asinfected leaves, the transcript level of PsMV2 was reduced by 80%, 41%, and 35% at 24, 48, and 120 hpi, respectively ( Figure 5D). These results indicated that the expression of PsMV2 was partially knocked down by HIGS, and silencing of PsMV2 reduced the pathogenicity and restricted the growth and development of Pst on wheat plants.

HIGS of PsMV2 Inhibits the Growth of Pst Mycelia and Increases the Accumulation of H 2 O 2 in Wheat Cells
To evaluate the histological responses associated with PsMV2 silencing, we analyzed hyphal length and infection areas at the infection site. At 24, 48, and 120 h after inoculation with Pst race CYR31 (strain GS-1), the histological changes in the pathogen were observed by microscopy and were estimated by DP-BSW software ( Figure 6A). The fluorescence microscope revealed that the hypha length and infected area were significantly less in BSMV: PsMV2 knock-down plants than in BSMV: γ-infected plants at 48 and 120 hpi ( Figure 6B,C).
The accumulation of reactive oxygen species and the occurrence of HR occur in the resistance of wheat in response to rust fungus [46]. To analyze the host response, we analyzed H 2 O 2 accumulation upon inoculation of wheat with Pst race CYR31 (strain GS-1) ( Figure 7A). The results showed that H 2 O 2 accumulation was expressively increased after silencing treated BSMV: PsMV2-1/2as leaves at 24 and 48 hpi ( Figure 7B).

Wide Distribution of PsMV2 in Wheat Stripe Rust
To detect the host range of the PsMV2, we performed RT-PCR for a range of plant and fungal samples (Figure 8). PsMV2 was detected in a wide range of Pst isolates differing in race and geographic location (Supplementary Table S4), as well as in P. graminis f. sp. tritici and P. recondita f. sp. tritici. On the other hand, PsMV2 was not detected in plant samples, including wheat, Mahonia fortune (Lindl.) Fedde, Berberis thunbergii DC, Hordeum vulgare L, and Brachypodium sylvaticum (Huds.) Beauv (Figure 8). These results indicate that PsMV2 might be a parasitic mycovirus widespread in wheat stripe rust fungus.
( Figure 5D). Similarly, in BSMV: PsMV2-2as-infected leaves, the transcript level of PsMV2 was reduced by 80%, 41%, and 35% at 24, 48, and 120 hpi, respectively ( Figure 5D). These results indicated that the expression of PsMV2 was partially knocked down by HIGS, and silencing of PsMV2 reduced the pathogenicity and restricted the growth and development of Pst on wheat plants.  hyphal length and infection areas at the infection site. At 24, 48, and 120 h after inoculation with Pst race CYR31 (strain GS-1), the histological changes in the pathogen were observed by microscopy and were estimated by DP-BSW software ( Figure 6A). The fluorescence microscope revealed that the hypha length and infected area were significantly less in BSMV: PsMV2 knock-down plants than in BSMV: γ-infected plants at 48 and 120 hpi (Figure 6B,C). The accumulation of reactive oxygen species and the occurrence of HR occur in the resistance of wheat in response to rust fungus [46]. To analyze the host response, we analyzed H2O2 accumulation upon inoculation of wheat with Pst race CYR31 (strain GS-1) ( Figure 7A). The results showed that H2O2 accumulation was expressively increased after silencing treated BSMV: PsMV2-1/2as leaves at 24 and 48 hpi ( Figure 7B).  The accumulation of reactive oxygen species and the occurrence of HR occur in the resistance of wheat in response to rust fungus [46]. To analyze the host response, we analyzed H2O2 accumulation upon inoculation of wheat with Pst race CYR31 (strain GS-1) ( Figure 7A). The results showed that H2O2 accumulation was expressively increased after silencing treated BSMV: PsMV2-1/2as leaves at 24 and 48 hpi ( Figure 7B). To detect the host range of the PsMV2, we performed RT-PCR for a range of plant and fungal samples (Figure 8). PsMV2 was detected in a wide range of Pst isolates differing in race and geographic location (Supplementary Table S4), as well as in P. graminis f. sp. tritici and P. recondita f. sp. tritici. On the other hand, PsMV2 was not detected in plant samples, including wheat, Mahonia fortune (Lindl.) Fedde, Berberis thunbergii DC, Hordeum vulgare L, and Brachypodium sylvaticum (Huds.) Beauv (Figure 8). These results indicate that PsMV2 might be a parasitic mycovirus widespread in wheat stripe rust fungus.

Discussion
In recent years, with the development of next-generation sequencing technologies, an increasing number of mycoviruses have been identified [4,16,52]. Studying mycovirus pathogenicity is important for a better understanding of the molecular biology of viruses [26,38,53]. In this study, we described the complete nucleotide sequence, genome organization and biological characteristics of a novel mycovirus PsMV2 in Pst, the causal agent of wheat stripe rust. Phylogenetic analyses of RdRp strongly supported the conclusion that PsMV2 represents a new member of the genus Unuamitovirus within the family Mitoviridae. In addition, PsMV2 can suppress programmed cell death (PCD) triggered by Bax. Using host-induced gene silencing (HIGS) to knockdown the expression of RdRp in

Discussion
In recent years, with the development of next-generation sequencing technologies, an increasing number of mycoviruses have been identified [4,16,52]. Studying mycovirus pathogenicity is important for a better understanding of the molecular biology of viruses [26,38,53]. In this study, we described the complete nucleotide sequence, genome organization and biological characteristics of a novel mycovirus PsMV2 in Pst, the causal agent of wheat stripe rust. Phylogenetic analyses of RdRp strongly supported the conclusion that PsMV2 represents a new member of the genus Unuamitovirus within the family Mitoviridae. In addition, PsMV2 can suppress programmed cell death (PCD) triggered by Bax. Using host-induced gene silencing (HIGS) to knockdown the expression of RdRp in PsMV2 leads to decreased virulence of Pst. These results demonstrate the positive effect of PsMV2 on Pst virulence. Moreover, we also found that PsMV2 is widely parasitic in rust fungus. This research expands our knowledge of the ecology and evolution of viruses in the family Mitoviridae and offers insights into the sustainable control of wheat rust disease.
Viruses of the family Mitoviridae are +ssRNA viruses, encoding an RdRp protein, and the viruses exist as RNA-RdRp nucleoprotein complexes [14]. Mitoviruses essentially replicate within the mitochondria of their hosts [20]. Consequently, as with many fungal and plant mitochondrial genomes, mitoviruses share A-U richness [14]. In the mitovirus genome, the UGA codon encodes the aa tryptophan, not a stop codon as it does in the universal genetic code [20]. Our results show that the genome of PsMV2 does have a high proportion of A+U (52.3%) ( Figure 1A). Another feature of mitovirus genomic RNA is the presence of stem-loop structures at both the 5 end and the 3 end, due to the presence of inverted complementary sequences at their termini [48], which is the situation for many mitoviruses found in various fungi, such as Fusarium oxysporum f. sp. dianthi mitovirus 1, Sclerotinia sclerotiorum mitovirus 1, and Melanconiella theae mitovirus 1 [17,22,26]. These structures may play an important role in a number of aspects of mitovirus replication, including the protection of naked ssRNA from degradation and as a recognition site for RdRp [48,54]. PsMV2 could be folded into potentially stable stem-loop structures ( Figure 1B). The long-term co-evolution of mitoviruses and their fungal hosts would lead to important sequence differences in the mitovirus genome [48]. According to the phylogenetic tree, some mitoviruses isolated from different fungal species (e.g., PsMV2 and CrMV1) were relatively closely related in terms of phylogeny. The different mitoviruses may have evolved to exist prior to the differentiation of their fungal hosts.
Studying mycovirus pathogenicity is important for a better understanding of the molecular biology of viruses [26,38,53]. Mycoviruses have different effects on fungi, ranging from inducing a cryptic state to increasing the capacity of the host to produce disease (hypervirulence) [27]. Although previously reported mitoviruses have little or no effects on their natural hosts, some mitoviruses are associated with hypovirulence to fungal hosts, making them a potential alternative method of biological control of plant diseases [55]. Pst is an obligate biotrophic fungus without a protoplast transformation system, making it difficult to obtain sufficient amounts of experimental materials from obligate host fungi, and it can cause asymptomatic infection. In fungi, RNA silencing (or RNA interference) is thought to serve primarily as a defense mechanism against invasive nucleic acids and viruses [56]. Antiviral RNA silencing is substantiated in fungi, e.g., Fusarium graminearum, Cryphonectria parasitica and Pyricularia oryzae [57][58][59]. In the present study, we used a BSMV-HIGS approach to determine the role of PsMV2 in the wheat-Pst interaction. In the normal condition, it was found that PsMV2 infecting Pst race CYR31 (strain GS-1) underwent significant proliferation during the initial infection of wheat seedlings (Figure 3). On the other hand, when Pst race CYR31 (strain GS-1) invaded wheat seedlings in which RNA silencing against PsMV2 is activated using HIGS, it was revealed that the amount of PsMV2 infecting Pst decreased, and subsequently, the disease symptoms, fungal biomass, and mycelial dispersal by Pst decreased compared to the normal condition ( Figure 5). These results suggested that PsMV2 is essential for the growth and development of Pst, which may be related to insufficient carbon and energy supply ( Figure 6). In addition, we found that PsMV2 might be a parasitic mycovirus widespread in field Pst isolates ( Figure 8 and Table S3) it is, therefore, possible that the gain of PsMV2 confers selective advantage (e.g., hypervirulence) and has been stabilized by Pst during evolution.
In summary, PsMV2 is a novel +ssRNA mitovirus identified from the fungus Pst. PsMV2 plays a positive role in the virulence of Pst. The detection of PsMV2 in wheat rust fungus obtained from 10 different countries indicates that PsMV2 has a wide geographical distribution in Pst. Future research will be focused on generating PsMV2-RNAi transgenic material and exploring whether it has broad-spectrum disease resistance, thus providing a new strategy against rust pathogens in wheat.

Supplementary Materials:
The following supporting information can be downloaded at: https://www. mdpi.com/article/10.3390/v15061265/s1, Table S1-list of the oligonucleotide primers used in this study; Table S2-sequence identities of PsMV2 to other viruses used for multiple sequence alignment and phylogenetic analysis; Table S3-Similarity analysis of PsMV2 to other viruses used for RNA-dependent RNA polymerase (RdRp); Table S4-RT-PCR detection of PsMV2 in 20 Pst samples collected in different regions in China (11 different provinces) and in 10 Pst samples from 10 other countries.