21Fusarium oxysporum is an economically important pathogen causing wilting or rotting 22 disease symptoms in a large number of crops. It is proposed to have a structured, 23 "two-speed" genome: i.e. regions containing genes involved in pathogenicity cluster 42 genes evolving at similar rates could increase evolvability. 43 44

Mitoviruses (genus Mitovirus, family Narnaviridae) are mitochondrially replicating viruses that have the simplest positive-sense RNA genomes of 2.2 to 4.4 kb with a single open reading frame (ORF) encoding an RNA-dependent RNA polymerase. Cryphonectria parasitica mitovirus 1 (CpMV1) from U.S. strain NB631 of the chestnut blight fungus, Cryphonectria parasitica, was the first virus identified as a mitochondrially replicating virus. Despite subsequent discovery of many other mitoviruses from diverse fungi, no great advances in understanding mitovirus biology have emerged, partly because of the lack of inoculation methods. Here we developed a protoplast fusion-based protocol for horizontal transmission of CpMV1 that entailed fusion of recipient and donor protoplasts, hyphal anastomosis, and single-conidium isolation. This method allowed expansion of the host range to many other C. parasitica strains. Species within and outside the family Cryphonectriaceae, Cryphonectria radicalis and Valsa ceratosperma, also supported the replication of CpMV1 at a level comparable to that in the natural host. No stable maintenance of CpMV1 was observed in Helminthosporium victoriae. PCR-based haplotyping of virus-infected fungal strains confirmed the recipient mitochondrial genetic background. Phenotypic comparison between CpMV1-free and -infected isogenic strains revealed no overt effects of the virus. Taking advantage of the infectivity to the standard strain C. parasitica EP155, accumulation levels were compared among antiviral RNA silencing-proficient and -deficient strains in the EP155 background. Comparable accumulation levels were observed among these strains, suggesting the avoidance of antiviral RNA silencing by CpMV1, which is consistent with its mitochondrial replication. Collectively, the results of study provide a foundation to further explore the biology of mitoviruses. IMPORTANCE Capsidless mitoviruses, which are ubiquitously detected in filamentous fungi, have the simplest RNA genomes of 2.2 to 4.4 kb, encoding only RNA-dependent RNA polymerase. Despite their simple genomes, detailed biological characterization of mitoviruses has been hampered by their mitochondrial location within the cell, posing challenges to their experimental introduction and study. Here we developed a protoplast fusion-based protocol for horizontal transfer of the prototype mitovirus, Cryphonectria parasitica mitovirus 1 (CpMV1), which was isolated from strain NB631 of the chestnut blight fungus (Cryphonectria parasitica), a model filamentous fungus for studying virus-host interactions. The host range of CpMV1 has been expanded to many different strains of C. parasitica and different fungal species within and outside the Cryphonectriaceae. Comparison of CpMV1 accumulation among various RNA silencing-deficient and -competent strains showed clearly that the virus was unaffected by RNA silencing. This study provides a solid foundation for further exploration of mitovirus-host interactions.

Recent studies have shown horizontal transfer of chromosomes to be a potential key contributor to genome plasticity in asexual fungal pathogens. However, the mechanisms behind horizontal chromosome transfer in eukaryotes are not well understood. Here we investigated the role of conidial anastomosis in heterokaryon formation between incompatible strains of Fusarium oxysporum and determined the importance of heterokaryons for horizontal chromosome transfer. Using live-cell imaging we demonstrate that conidial pairing of incompatible strains under carbon starvation can result in the formation of viable heterokaryotic hyphae in F. oxysporum. Nuclei of the parental lines presumably fuse at some stage as conidia with a single nucleus harboring both marker histones (GFP- and RFP-tagged) are produced. Upon colony formation, this hybrid offspring is subject to progressive and gradual genome rearrangement. The parental genomes appear to become spatially separated and RFP-tagged histones, deriving from one of the strains, Fol4287, are eventually lost. With a PCR-based method we showed that markers for most of the chromosomes of this strain are lost, indicating a lack of Fol4287 chromosomes. This leaves offspring with the genomic background of the other strain (Fo47), but in some cases together with one or two chromosomes from Fol4287, including the chromosome that confers pathogenicity towards tomato.

Root architecture and growth patterns are plant features that are still poorly understood. When grown under in vitro conditions, seedlings with mutations in Arabidopsis thaliana genes MLO4 or MLO11 exhibit aberrant root growth patterns upon contact with hard surfaces, exemplified as tight root spirals. We used a set of physiological assays and genetic tools to characterize this thigmomorphogenic defect in detail. We observed that the mlo4/mlo11-associated root curling phenotype is not recapitulated in a set of mutants with altered root growth patterns or architecture. We further found that mlo4/mlo11-conditioned root curling is not dependent upon light and endogenous flavonoids, but is pH-sensitive and affected by exogenous calcium levels. Based upon the latter two characteristics, mlo4-associated root coiling appears to be mechanistically different from the natural strong root curvature of the Arabidopsis ecotype Landsberg erecta. Gravistimulation reversibly overrides the aberrant thigmomorphogenesis of mlo4 seedlings. Mutants with dominant negative defects in α-tubulin modulate the extent and directionality of mlo4/mlo11-conditioned root coils, whereas mutants defective in polar auxin transport (axr4, aux1) or gravitropism (pgm1) completely suppress the mlo4 root curling phenotype. Our data implicate a joint contribution of calcium signalling, pH regulation, microtubular function, polar auxin transport and gravitropism in root thigmomorphogenesis.

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