Completion of the genome sequence of Oidiodendron maius splipalmivirus 1.

Mycoviruses with an unprecedented genome organization, featuring the RNA-directed RNA polymerase (RdRp) palm domain coding sequence being split into two distinct genome segments, have been found recently in a few fungi and oomycetes of different lineages and have been proposed to be named "splipalmiviruses". One of these, Oidiodendron maius splipalmivirus 1 (OmSPV1), has been detected in the ericoid mycorrhizal fungus Oidiodendron maius, and it has been proposed to be bisegmented. Here, we complete the genome sequence of this virus by describing a third RNA segment, which is 2000 nt long and whose terminal sequences are identical to those of the other two segments of OmSPV1. This segment contains a single open reading frame that codes for a protein with unknown function and has a low level of sequence identity (47%) to the putative protein encoded by the third segment of another splipalmivirus from Magnaporthe oryzae: Magnaporthe oryzae narnavirus virus 1 (MoNV1). Based on these features, we propose the RNA segment to be the third segment of the OmSPV1 genome.

Complete Genome Analyses of a Novel Flexivirus with Unique Genome Organization and Three Endornaviruses Hosted by the Mycorrhizal Fungus Terfezia claveryi.

The extensive use of high-throughput sequencing (HTS) has significantly advanced and transformed our comprehension of virus diversity, especially in intricate settings like soil and biological specimens. In this study, we delved into mycovirus sequence surveys within mycorrhizal fungus species Terfezia claveryi, through employing HTS with total double-stranded RNA (dsRNA) extracts. Our findings revealed the presence of four distinct members from the Alsuviricetes class, one flexivirus designated as Terfezia claveryi flexivirus 1 (TcFV1) and three endornaviruses (TcEV1, TcEV2, and TcEV3) in two different T. claveryi isolates. TcFV1, a member of the order Tymovirales, exhibits a unique genome structure and sequence features. Through in-depth analyses, we found that it shares sequence similarities with other deltaflexiviruses and challenges existing Deltaflexiviridae classification. The discovery of TcFV1 adds to the genomic plasticity of mycoviruses within the Tymovirales order, shedding light on their evolutionary adaptations. Additionally, the three newly discovered endornaviruses (TcEV1, TcEV2, and TcEV3) in T. claveryi exhibited limited sequence similarities with other endornaviruses and distinctive features, including conserved domains like DEAD-like helicase, ATPases Associated with Diverse Cellular Activities (AAA ATPase), and RNA dependent RNA polymerase (RdRp), indicating their classification as members of new species within the Alphaendornavirus genus. In conclusion, this research emphasizes the importance of exploring viral diversity in uncultivated fungi, bridging knowledge gaps in mycovirus ecology. The discoveries of a novel flexivirus with unique genome organization and endornaviruses in T. claveryi broaden our comprehension of mycovirus diversity and evolution, highlighting the need for continued investigations into viral populations in wild fungi.

Virome analysis of an ectomycorrhizal fungus Suillus luteus revealing potential evolutionary implications.

Suillus luteus is a widespread edible ectomycorrhizal fungus that holds significant importance in both ecological and economic value. Mycoviruses are ubiquitous infectious agents hosted in different fungi, with some known to exert beneficial or detrimental effects on their hosts. However, mycoviruses hosted in ectomycorrhizal fungi remain poorly studied. To address this gap in knowledge, we employed next-generation sequencing (NGS) to investigate the virome of S. luteus. Using BLASTp analysis and phylogenetic tree construction, we identified 33 mycovirus species, with over half of them belonging to the phylum Lenarviricota, and 29 of these viruses were novel. These mycoviruses were further grouped into 11 lineages, with the discovery of a new negative-sense single-stranded RNA viral family in the order Bunyavirales. In addition, our findings suggest the occurrence of cross-species transmission (CST) between the fungus and ticks, shedding light on potential evolutionary events that have shaped the viral community in different hosts. This study is not only the first study to characterize mycoviruses in S. luteus but highlights the enormous diversity of mycoviruses and their implications for virus evolution.

Omnipresence of Partitiviruses in Rice Aggregate Sheath Spot Symptom-Associated Fungal Isolates from Paddies in Thailand.

Partitiviruses are one of the most prevalent double-stranded RNA viruses that have been identified mostly in filamentous fungi and plants. Partitiviruses generally infect host fungi asymptomatically but infrequently exert significant effect(s) on morphology and virulence, thus being considered a potential source of biological control agents against pathogenic fungi. In this study, we performed a screening for mycoviruses of a collection of Thai isolates of rice fungal pathogen Rhizoctonia oryzae-sativae, a causal agent of rice aggregated sheath spot disease. As a result, 36% of tested isolates carried potentially viral double-stranded RNAs with sizes ranging from 2 to 3 kbp. By conventional cDNA library construction and RNA-seq, we determined six new alphapartitiviruses that infected three isolates: tentatively named Rhizoctonia oryzae-sativae partitivirus 1 to 6 (RosPV1-6). Furthermore, RT-PCR detection of each virus revealed their omnipresent nature in different R. oryzae-sativae isolates. Although virus-curing of basidiomycetous fungi is generally difficult, our repeated attempts successfully obtained virus-free (for RosPV1, RosPV2, and uncharacterized partitiviruses), isogenic strain of R. oryzae-sativae TSS190442. The virus-cured strain showed slightly faster colony growth on the synthetic media and severe symptom development on the rice sheath compared to its virus-infected counterpart. Overall, this study shed light on the distribution of partitiviruses in R. oryzae-sativae in a paddy environment and exemplified a virus-curing protocol that may be applicable for other basidiomycetous fungi.

Molecular characterization of a novel fusarivirus infecting the edible fungus Auricularia heimuer.

Here, we describe a novel mycovirus, Auricularia heimuer fusarivirus 1 (AhFV1), isolated from the edible fungus Auricularia heimuer strain CCMJ1296. The virus has a single-stranded positive-sense [+ssRNA] genome of 7,127 nucleotides containing two overlapping open reading frames (ORFs) and a poly(A) tail. The large ORF1 encodes a polyprotein of 1,637 amino acids (aa) with conserved RNA-dependent RNA polymerase (RdRp) and DEAD-like helicase superfamily (DEXDc) domains. ORF2 encodes a putative 633-aa protein with unknown function. A BLAST search showed that the nucleotide sequence of the AhFV1 genome is 41.28% identical to that of Sclerotium rolfsii fusarivirus 2 and 40.49% identical to that of Sclerotium rolfsii fusarivirus 1. Phylogenetic analysis based on RdRp and helicase (Hel) sequences indicated that AhFV1 is related to unclassified mycoviruses and other fusariviruses. Our data suggest that AhFV1 should be classified as a member of the newly proposed family "Fusariviridae". This is the second virus and the first full genome sequence of a fusarivirus from A. heimuer.

Mixed infection by a partitivirus and a negative-sense RNA virus related to mymonaviruses in the polypore fungus Bondarzewia berkeleyi.

Virus communities of forest fungi remain poorly characterized. In this study, we detected two new viruses co-infecting an isolate of the polypore fungus Bondarzewia berkeleyi using high-throughput sequencing. One of them was a putative new partitivirus designated as Bondarzewia berkeleyi partitivirus 1 (BbPV1), with two linear dsRNA genome segments of 1928 and 1863 bp encoding a putative RNA-dependent RNA polymerase (RdRP) of 591 aa and a putative capsid protein of 538 aa. The other virus, designated as Bondarzewia berkeleyi negative-strand RNA virus 1 (BbNSRV1), had a non-segmented negative-sense RNA genome of 10,983 nt and was related to members of family Mymonaviridae. The BbNSRV1 genome includes six predicted open reading frames (ORFs) of 279, 425, 230, 174, 200 and 1970 aa. The longest ORF contained conserved regions corresponding to Mononegavirales RdRP and mRNA-capping enzyme region V constituting the mononegavirus Large protein. In addition, a low level of sequence identity was detected between the putative nucleocapsid protein-coding ORF2 of Lentinula edodes negative-strand RNA virus 1 and BbNSRV1. The viruses characterized in this study are the first ones described in Bondarzewia spp., and BbNSRV1 is the second mymona-like virus described in a basidiomycete host.

Virus population structure in the ectomycorrhizal fungi Lactarius rufus and L. tabidus at two forest sites in Southern Finland.

Lactarius fungi belong to the Russulaceae family and have an important ecological role as ectomycorrhizal symbionts of coniferous and deciduous trees. Two Lactarius species, L. tabidus and L. rufus have been shown to harbor bisegmented dsRNA viruses belonging to an unclassified virus group including the mutualistic Curvularia thermal tolerance virus (CThTV). In this study, we characterized the first complete genome sequences of these viruses designated as Lactarius tabidus RNA virus 1 (LtRV1) and Lactarius rufus RNA virus 1 (LrRV1), both of which included two genome segments of 2241 and 2049 bp. We also analyzed spatial distribution and sequence diversity of the viruses in sixty host strains at two forest sites, and showed that the viruses are species-specific at sites where both host species co-occur. We also found that single virus isolates inhabited several different conspecific host strains, and were involved in persistent infections during up to eight years.

Molecular characteristics of a novel ssRNA virus isolated from Auricularia heimuer in China.

A novel positive-sense single-stranded RNA virus was isolated from strain CCMJ1271 of the fungus Auricularia heimuer, and the complete genome sequence of the virus was determined. Database searching, sequence alignment, and phylogenetic analysis revealed that this fungal virus and some viruses of family Virgaviridae clustered into a single branch of a phylogenetic tree, and we thus tentatively named the virus "Auricularia heimuer mycovirgavirus 1" (AhMV1). The AhMV1 genome consists of 9,934 nucleotides and contains a short poly(A) tail and three open reading frames (ORFs). ORF1 encodes an RNA-dependent RNA polymerase (RdRp), ORF2 encodes a protein that is homologous to movement proteins of plant virgaviruses, and ORF3 encodes a coat protein (CP). AhMV1 is the first virus to be discovered in A. heimuer.

A novel narnavirus isolated from the wheat stripe rust fungus Puccinia striiformis f. sp. tritici.

The complete genome of a novel fungal virus, Puccinia striiformis narnavirus 1 (PsNV1), was sequenced and analyzed. The full-length cDNA sequence is 2340 bp in length with a GC content of 50.04%. PsNV1 contains a single open reading frame (ORF), which encodes a putative RNA-dependent RNA polymerase (RdRp) of 741 amino acids with a molecular mass of 81.8 kDa. RdRp phylogeny showed that PsNV1 grouped together with Fusarium poae narnavirus 1 (FpNV1) as a sister branch of narnaviruses, forming a distinct clade. The results of genome sequence comparisons and phylogenetic analysis indicate that PsNV1 is a new member in the genus Narnavirus. To our knowledge, this is the first report of a narnavirus genome sequence in the obligately parasitic fungus Puccinia striiformis f. sp. tritici.

Multiple virus infections on Heterobasidion sp.

Heterobasidion viruses have previously been shown to affect each other's transmission and phenotypic effects on their hosts in a complex way. In this work, Heterobasidion parviporum strains hosting five coinfecting viruses simultaneously were constructed and used as donors in transmission experiments. They showed that viruses move more frequently between the mycelia of the same species than between the mycelia of H. parviporum and Heterobasidionannosum. One of the strains was used to show that coinfection of five viruses is relatively unstable in a natural environment and analyses of the growth rate and competitive ability of Heterobasidion strains hosting various virus combinations revealed that viral effects are not additive. The results also supported the view that the transmission of the promising virocontrol agent HetPV13-an1 may be enhanced by coinfecting viruses in the donor mycelium. However, its detrimental effects may be blocked by the presence of other viruses in the same mycelium. REPOSITORIES: GenBank accession number MN058080.

Characterization of Cronartium ribicola dsRNAs reveals novel members of the family Totiviridae and viral association with fungal virulence.

BACKGROUND: Mycoviruses were recently discovered in the white pine blister rust (WPBR) fungus Cronartium ribicola (J.C. Fisch.). Detection and characterization of their double stranded RNA (dsRNA) would facilitate understanding of pathogen virulence and disease pathogenesis in WPBR systems. METHODS: Full-length cDNAs were cloned from the dsRNAs purified from viral-infected C. ribicola, and their cDNA sequences were determined by DNA sequencing. Evolutionary relationships of the dsRNAs with related mycoviruses were determined by phylogenetic analysis. Dynamic distributions of the viral RNAs within samples of their fungal host C. ribicola were investigated by measurement of viral genome prevalence and viral gene expression. RESULTS: In this study we identified and characterized five novel dsRNAs from C. ribicola, designated as Cronartium ribicola totivirus 1-5 (CrTV1 to CrTV5). These dsRNA sequences encode capsid protein and RNA-dependent RNA polymerase with significant homologies to dsRNA viruses of the family Totiviridae. Phylogenetic analysis showed that the CrTVs were grouped into two distinct clades. CrTV2 through CrTV5 clustered within the genus Totivirus. CrTV1 along with a few un-assigned dsRNAs constituted a distinct phyletic clade that is genetically distant from presently known genera in the Totiviridae family, indicating that CrTV1 represents a novel genus in the Totiviridae family. The CrTVs were prevalent in fungal samples obtained from infected western white pine, whitebark pine, and limber pines. Viral RNAs were generally expressed at higher levels during in planta mycelium growth than in aeciospores and urediniospores. CrTV4 was significantly associated with C. ribicola virulent pathotype and specific C. ribicola host tree species, suggesting dsRNAs as potential tools for dissection of pathogenic mechanisms of C. ribicola and diagnosis of C. ribicola pathotypes. CONCLUSION: Phylogenetic and expression analyses of viruses in the WPBR pathogen, C. ribicola, have enchanced our understanding of virus diversity in the family Totiviridae, and provided a potential strategy to utilize pathotype-associated mycoviruses to control fungal forest diseases.

Challenges to elucidating how endornaviruses influence fungal hosts: Creating mycovirus-free isogenic fungal lines and testing them.

Determining roles of mycoviruses in fungal biology is complicated, especially when fungi are co-infected with multiple viruses. Genetically identical (isogenic) fungal lines that are infected by and not infected by viruses must be created and compared. Here, we study an isolate of Ceratobasidium sp., a fungus isolated from pelotons in roots of a wild terrestrial orchid. The fungal isolate was co-infected with three distinct endornaviruses, isolates of Ceratobasidium endonarvirus B (CbEVB), Ceratobasidium endonarvirus C (CbEVC) and Ceratobasidium endonarvirus D (CbEVD). An experiment to reveal natural distribution of the three mycoviruses within a fungal colony revealed no sectoring; they were all evenly distributed throughout the colony. Hyphal tipping and treatments with one of five antibiotics (kanamycin, streptomycin, cycloheximide, rifampicin and ampicillin) were applied in attempts to 'cure' fungal lines of one, two or three of the viruses present. Surprisingly, the three mycoviruses responded differentially to each curing approach. The isolate of CbEVC was eliminated upon treatment with cycloheximide, but not with kanamycin or streptomycin, whereas the isolate of CbEVD did not respond to cycloheximide. The isolate of CbEVB was eliminated upon all treatments. In some cases, a virus was undetectable by species-specific RT-PCR assay after treatment, but when the fungus was cultured for a period on non-selective medium, the virus was detected again. Effects of mycoviruses on growth characteristics of isogenic fungal lines on two nutrient media were studied. Co-infection by the three viruses reduced mycelial growth rate on both media. In contrast, some fungal lines infected with one or two mycoviruses grew more rapidly than virus-free lines.

Mitoviruses in the conifer root rot pathogens Heterobasidion annosum and H. parviporum.

Mitoviral infections are highly common among fungi, but so far only one mitovirus has been described in Heterobasidion spp. conifer pathogens. Here, the occurrence of further mitoviruses was investigated using a previously published RNA-Seq dataset for de novo contig assembly. This allowed the identification of two additional mitovirus strains designated as Heterobasidion mitovirus 2 (HetMV2) and HetMV3 with genome lengths of ca. 2.9 and 5.0 kb. Furthermore, the occurrence of similar viruses was screened among a collection of Heterobasidion isolates using RT-PCR. Mitoviruses were detected in six more fungal isolates and two different host species, H. annosum and H. parviporum.

Alphapartitiviruses of Heterobasidion Wood Decay Fungi Affect Each Other's Transmission and Host Growth.

Heterobasidion spp. root rot fungi are highly destructive forest pathogens of the northern boreal forests, and are known to host a diverse community of partitiviruses. The transmission of these mycoviruses occurs horizontally among host strains via mycelial anastomoses. We revealed using dual cultures that virus transmission rates are affected by pre-existing virus infections among two strains of H. annosum. The transmission efficacy of mycovirus HetPV15-pa1 to a pre-infected host was elevated from zero to 50% by the presence of HetPV13-an1, and a double infection of these viruses in the donor resulted in an overall transmission rate of 90% to a partitivirus-free recipient. On contrary, pre-existing virus infections of two closely related strains of HetPV11 hindered each other's transmission, but had unexpectedly dissimilar effects on the transmission of more distantly related viruses. The co-infection of HetPV13-an1 and HetPV15-pa1 significantly reduced host growth, whereas double infections including HetPV11 strains had variable effects. Moreover, the results showed that RdRp transcripts are generally more abundant than capsid protein (CP) transcripts and the four different virus strains express unique transcripts ratios of RdRp and CP. Taken together, the results show that the interplay between co-infecting viruses and their host is extremely complex and highly unpredictable.

Complete genome sequence of a novel mitovirus from the wheat stripe rust fungus Puccinia striiformis.

The complete genome of a novel mycovirus, Puccinia striiformis mitovirus 1 (PsMV1), derived from the wheat stripe rust fungus Puccinia striiformis strain SCSN-10, was sequenced and analyzed. The full-length cDNA sequence is 2496 bp in length with a predicted AU content of 57.65% in the genomic RNA. Sequence analysis indicated that a single large open reading frame (ORF) is present on the positive strand when the fungal mitochondrial genetic code is used. The single ORF encodes a putative RNA-dependent RNA polymerase of 743 amino acids with a molecular mass of 84.9 kDa that shares the closest similarity with the corresponding proteins of Cronartium ribicola mitovirus 5 and Helicobasidium mompa mitovirus 1-18 (34% and 35% aa sequence identity, respectively). Phylogenetic analysis further indicated that PsMV1 is a new member of the genus Mitovirus within the family Narnaviridae. This is the first report of the full-length nucleotide sequence of a novel mitovirus, PsMV1, from the causal agent of wheat stripe rust.

The complete genome sequence of HetPV20-an1, an alphapartitivirus infecting the conifer-pathogenic fungus Heterobasidion annosum.

Root rot fungi of the genus Heterobasidion are highly destructive conifer pathogens in the northern Boreal forest region. This report describes the complete genome sequence of Heterobasidion partitivirus 20 infecting a Finnish strain of Heterobasidion annosum. The bisegmented dsRNA genome of HetPV20-an1 encodes a predicted RNA-dependent RNA polymerase of 605 amino acids (aa) and a capsid protein of 536 aa. Based on sequence similarity and phylogenetic analysis, this virus is a new member of the genus Alphapartitivirus. HetPV20-an1 shares ~65% RdRP aa sequence identity with the most similar virus strain, Rosellinia necatrix partitivirus 2, whereas the CP of HetPV20-an1 is most similar to that of rose partitivirus with ~27% overall aa sequence identity. HetPV20-an1 is only distantly related to previously known partitiviruses of Heterobasidion species and shares ~29% RdRP aa sequence identity and ~16% CP aa sequence identity with Heterobasidion partitivirus 1 from H. abietinum.

Heterobasidion Partitivirus 13 Mediates Severe Growth Debilitation and Major Alterations in the Gene Expression of a Fungal Forest Pathogen.

The fungal genus Heterobasidion includes some of the most devastating conifer pathogens in the boreal forest region. In this study, we showed that the alphapartitivirus Heterobasidion partitivirus 13 from Heterobasidion annosum (HetPV13-an1) is the main causal agent of severe phenotypic debilitation in the host fungus. Based on RNA sequencing using isogenic virus-infected and cured fungal strains, HetPV13-an1 affected the transcription of 683 genes, of which 60% were downregulated and 40% upregulated. Alterations observed in carbohydrate and amino acid metabolism suggest that the virus causes a state of starvation, which is compensated for by alternative synthesis routes. We used dual cultures to transmit HetPV13-an1 into new strains of H. annosum and Heterobasidion parviporum The three strains of H. parviporum that acquired the virus showed noticeable growth reduction on rich culturing medium, while only two of six H. annosum isolates tested showed significant debilitation. Based on reverse transcription-quantitative PCR (RT-qPCR) analysis, the response toward HetPV13-an1 infection was somewhat different in H. annosum and H. parviporum We assessed the effects of HetPV13-an1 on the wood colonization efficacy of H. parviporum in a field experiment where 46 Norway spruce trees were inoculated with isogenic strains with or without the virus. The virus-infected H. parviporum strain showed considerably less growth within living trees than the isolate without HetPV13-an1, indicating that the virus also causes growth debilitation in natural substrates.IMPORTANCE A biocontrol method restricting the spread of Heterobasidion species would be highly beneficial to forestry, as these fungi are difficult to eradicate from diseased forest stands and cause approximate annual losses of €800 million in Europe. We used virus curing and reintroduction experiments and RNA sequencing to show that the alphapartitivirus HetPV13-an1 affects many basic cellular functions of the white rot wood decay fungus Heterobasidion annosum, which results in aberrant hyphal morphology and a low growth rate. Dual fungal cultures were used to introduce HetPV13-an1 into a new host species, Heterobasidion parviporum, and field experiments confirmed the capability of the virus to reduce the growth of H. parviporum in living spruce wood. Taken together, our results suggest that HetPV13-an1 shows potential for the development of a future biocontrol agent against Heterobasidion fungi.

Distribution of Viruses Inhabiting Heterobasidion annosum in a Pine-Dominated Forest Plot in Southern Finland.

We investigated the diversity and spatial distribution of viruses infecting strains of the root rot fungus Heterobasidion annosum collected from pine stumps at a heavily infected forest site. Four different partitiviruses were detected in 14 H. annosum isolates at the study site, constituting approximately 29% of all Heterobasidion isolates investigated (N = 48). Two of the viruses detected were new partitiviruses designated here as Heterobasidion partitivirus 16 (HetPV16) and HetPV20, and two were previously known partitiviruses: HetPV7 and HetPV13. The two new partitiviruses found, HetPV16-an1 and HetPV20-an1, shared ~70% RdRp nucleotide sequence identity with the alphapartitivirus Rosellinia necatrix partitivirus 2, and less than 40% identity with known viruses of Heterobasidion spp. HetPV7-an1 was closely similar to HetPV7-pa1 isolated earlier from Heterobasidion parviporum, supporting the view of conspecific virus pools in different Heterobasidion species. Three fungal isolates were found to be co-infected with two different partitivirus strains (HetPV7-an1 and HetPV13-an2 or HetPV16-an1 and HetPV20-an1). Different isolates representing each host clone had variable virus compositions, and virus strains occurring in more than one host clone showed minor sequence variations between clones.

ICTV Virus Taxonomy Profile: Chrysoviridae.

The Chrysoviridae is a family of small, isometric, non-enveloped viruses (40 nm in diameter) with segmented dsRNA genomes (typically four segments). The genome segments are individually encapsidated and together comprise 11.5-12.8 kbp. The single genus Chrysovirus includes nine species. Chrysoviruses lack an extracellular phase to their life cycle; they are transmitted via intracellular routes within an individual during hyphal growth, in asexual or sexual spores, or between individuals via hyphal anastomosis. There are no known natural vectors for chrysoviruses. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the Chrysoviridae, which is available at www.ictv.global/report/chrysoviridae.

Investigating Gene Function in Cereal Rust Fungi by Plant-Mediated Virus-Induced Gene Silencing.

Cereal rust fungi are destructive pathogens, threatening grain production worldwide. Targeted breeding for resistance utilizing host resistance genes has been effective. However, breakdown of resistance occurs frequently and continued efforts are needed to understand how these fungi overcome resistance and to expand the range of available resistance genes. Whole genome sequencing, transcriptomic and proteomic studies followed by genome-wide computational and comparative analyses have identified large repertoire of genes in rust fungi among which are candidates predicted to code for pathogenicity and virulence factors. Some of these genes represent defence triggering avirulence effectors. However, functions of most genes still needs to be assessed to understand the biology of these obligate biotrophic pathogens. Since genetic manipulations such as gene deletion and genetic transformation are not yet feasible in rust fungi, performing functional gene studies is challenging. Recently, Host-induced gene silencing (HIGS) has emerged as a useful tool to characterize gene function in rust fungi while infecting and growing in host plants. We utilized Barley stripe mosaic virus-mediated virus induced gene silencing (BSMV-VIGS) to induce HIGS of candidate rust fungal genes in the wheat host to determine their role in plant-fungal interactions. Here, we describe the methods for using BSMV-VIGS in wheat for functional genomics study in cereal rust fungi.