Mycovirus-infected fungi can suffer from poor growth, attenuated pigmentation, and virulence. However, the molecular mechanisms of how mycoviruses confer these symptoms remain poorly understood. Here, we report a mycovirus Stemphylium lycopersici alternavirus 1 (SlAV1) isolated from a necrotrophic plant pathogen Stemphylium lycopersici that causes altered colony pigmentation and hypovirulence by specifically interfering host biosynthesis of Altersolanol A, a polyketide phytotoxin. SlAV1 significantly down-regulates a fungal polyketide synthase ( PKS1 ), the core enzyme of Altersolanol A biosynthesis. PKS1 deletion mutants do not accumulate Altersolanol A and lose pathogenicity to tomato and lettuce. Transgenic expression of SlAV1 open-reading frame 3 (ORF3) in S. lycopersici inhibits fungal PKS1 expression and Altersolanol A accumulation, leading to symptoms like SlAV1-infected fungal strains. Multiple plant species sprayed with mycelial suspension of S. lycopersici or S. vesicarium strains integrating and expressing ORF3 display enhanced resistance against virulent strains, converting the pathogenic fungi into biocontrol agents. Hence, our study not only proves inhibiting a key enzyme of host phytotoxin biosynthesis as a molecular mechanism underlying SlAV1-mediated hypovirulence of Stemphylium spp., but also demonstrates the potential of mycovirus-gene integrated fungi as a potential biocontrol agent to protect plants from fungal diseases.

The Pseudomonas syringae effector protein AvrRpm1 activates the Arabidopsis (Arabidopsis thaliana) intracellular innate immune receptor protein RESISTANCE TO PSEUDOMONAS MACULICOLA1 (RPM1) via modification of a second Arabidopsis protein, RPM1-INTERACTING PROTEIN4 (AtRIN4). Prior work has shown that AvrRpm1 induces phosphorylation of AtRIN4, but homology modeling indicated that AvrRpm1 may be an ADP-ribosyl transferase. Here, we show that AvrRpm1 induces ADP-ribosylation of RIN4 proteins from both Arabidopsis and soybean (Glycine max) within two highly conserved nitrate-induced (NOI) domains. It also ADP ribosylates at least 10 additional Arabidopsis NOI domain-containing proteins. The ADP-ribosylation activity of AvrRpm1 is required for subsequent phosphorylation on Thr-166 of AtRIN4, an event that is necessary and sufficient for RPM1 activation. We also show that the C-terminal NOI domain of AtRIN4 interacts with the exocyst subunits EXO70B1, EXO70E1, EXO70E2, and EXO70F1. Mutation of either EXO70B1 or EXO70E2 inhibited secretion of callose induced by the bacterial flagellin-derived peptide flg22. Substitution of RIN4 Thr-166 with Asp enhanced the association of AtRIN4 with EXO70E2, which we posit inhibits its callose deposition function. Collectively, these data indicate that AvrRpm1 ADP-ribosyl transferase activity contributes to virulence by promoting phosphorylation of RIN4 Thr-166, which inhibits the secretion of defense compounds by promoting the inhibitory association of RIN4 with EXO70 proteins.

Sclerotium rolfsii, which causes southern blight in a wide variety of crops, is a devastating plant pathogen worldwide. Mycoviruses that induce hypovirulence in phytopathogenic fungi are potential biological control resources against fungal plant diseases. However, in S. rolfsii, mycoviruses are rarely reported. In a previous study, we found a hypovirulent strain carrying a diverse pattern of dsRNAs. Here, we utilized the RNA_Seq technique to detect viral sequences. Deep sequencing, RT-PCR and Sanger sequencing validation analyses revealed that this strain harbors various new viral species that show affinity to the distinctly established and proposed families Benyviridae, Endornaviridae, Fusariviridae, Hypoviridae, and Fusagraviridae. Moreover, some viral sequences that could not be assigned to any of the existing families or orders were also identified and showed similarities to the Alphavirus, Ourmiavirus, phlegivirus-like and Curvularia thermal tolerance virus-like groups. In addition, we also conducted deep sequencing analysis of small RNAs in the virus-infecting fugal strain. The results indicated that the Dicer-mediated gene silencing mechanism was present in S. rolfsii. This is the first report of viral diversity in a single S. rolfsii fungal strain, and the results presented herein might provide insight into the taxonomy and evolution of mycoviruses and be useful for the exploration of mycoviruses as biocontrol agents.

Here, the molecular characterization of a novel mycovirus that was isolated from a phytopathogenic fungus Magnaporthe oryzae and designed as Magnaporthe oryzae ourmia-like virus 4 (MOLV4) is reported. MOLV4 has a genome that is 2497 bp long and possesses a single open reading frame (ORF), which encodes the product RNA-dependent RNA polymerase (RdRp). Sequence similarities were found between the MOLV4 encoded RdRp and the counterparts of a few previously reported ourmia-like mycoviruses. Virus-curing and biological comparison indicate that the virus has no or mild effects on the morphology and mycelium growth rate of the host fungus. Phylogenetic analysis using the RdRp aa sequences was performed. The results show that MOLV4 is clustered with the ourmia-like mycoviruses, forming a clade closely related to ourmiaviruses but distinct from narnaviruses. In addition, database searches revealed that several MOLV4-related sequences are present in the transcriptome shotgun assembly (TSA) library, expressed sequence tag database (ESTdb), whole-genome shotgun (WGS) library, and genomic survey sequences (GSS) libraries of a few other species of eukaryote organisms. Our results show that MOLV4, together with other similar ourmia-like mycoviruses, might represent a virus clade that links the plant ourmiaviruses and fungal narnaviruses and has a wide range of hosts.

Mycoviruses associated with hypovirulence are potential biological control agents and could be useful to study the pathogenesis of fungal host pathogens. Sclerotium rolfsii, a pathogenic fungus, causes southern blight in a wide variety of crops. In this study, we isolated a series of dsRNAs from a debilitated S. rolfsii strain, BLH-1, which had pronounced phenotypic aberrations including reduced pathogenicity, mycelial growth and deficient sclerotia production. Virus-curing and horizontal transmission experiments that eliminated or transmitted, respectively, all dsRNA elements showed that the dsRNAs were involved in the hypovirulent traits of BLH-1. Ultrastructure examination also showed hyphae fracture and cytoplasm or organelle degeneration in BLH-1 hyphal cells compared to the virus-free strain. Three assembled cDNA contigs generated from the cDNA library cloned from the purified dsRNA indicated that strain BLH-1 was infected by at least three novel mycoviruses. One has similarity to the hypovirulence-associated Sclerotinia sclerotiorum hypovirus 2 (SsHV2) in the family Hypoviridae, and the other two are related to two different unclassified dsRNA mycovirus families. To our knowledge, this is the first report of S. rolfsii hypovirulence that was correlated with its associated dsRNA.

Three dsRNAs, in sizes of approximately 2.5–5 kbp, were detected in the plant pathogenic fungus Nigrospora oryzae strain CS-7.5-4. Genomic analysis showed that the 5.0 kb dsRNA was a victorivirus named as Nigrospora oryzae victorivirus 2 (NoRV2). The genome of NoRV2 was 5166 bp in length containing two overlapping open reading frames (ORFs), ORF1 and ORF2. ORF1 was deduced to encode a coat protein (CP) showing homology to the CPs of viruses belonging to the Totiviridae family. The stop codon of ORF1 and the start codon of ORF2 were overlapped by the tetranucleotide sequence AUGA. ORF2 was predicted to encode an RNA-dependent RNA polymerase (RdRp), which was highly similar to the RdRps of victoriviruses. Virus-like particle examination demonstrated that the genome of NoRV2 was solely encapsidated by viral particles with a diameter of approximately 35 nm. The other two dsRNAs that were less than 3.0 kb were predicted to be the genomes of two mitoviruses, named as Nigrospora oryzae mitovirus 1 (NoMV1) and Nigrospora oryzae mitovirus 2 (NoMV2). Both NoMV1 and NoMV2 were A-U rich and with lengths of 2865 and 2507 bp, respectively. Mitochondrial codon usage inferred that each of the two mitoviruses contains a major large ORF encoding a mitoviral RdRp. Horizontal transfer experiments showed that the NoMV1 and NoMV2 could be cotransmitted horizontally via hyphal contact to other virus-free N. oryzae strains and causes phenotypic change to the recipient, such as an increase in growth rate. This is the first report of mitoviruses in N. oryzae.