Mycoviruses are wide spread throughout almost all groups of fungi but only a small number of mycoviruses can attenuate the growth and virulence of their fungal hosts. Alternaria alternata is an ascomycete fungus that causes leaf spot diseases on various crop plants. In this study, we identified a novel ssRNA mycovirus infecting an A. alternata f. sp. mali strain isolated from an apple orchard in China. Sequence analyses revealed that this virus is related to hypoviruses, in particular to Wuhan insect virus 14, an unclassified hypovirus identified from insect meta-transcriptomics, as well as other hypoviruses belonging to the genus Hypovirus , and therefore this virus is designed as Alternaria alternata hypovirus 1 (AaHV1). The genome of AaHV1 contains a single large open-reading frame encoding a putative polyprotein (∼479 kDa) with a cysteine proteinase-like and replication-associated domains. Curing AaHV1 from the fungal host strain indicated that the virus is responsible for the slow growth and reduced virulence of the host. AaHV1 defective RNA (D-RNA) with internal deletions emerging during fungal subcultures but the presence of D-RNA does not affect AaHV1 accumulation and pathogenicities. Moreover, AaHV1 could replicate and confer hypovirulence in Botryosphaeria dothidea , a fungal pathogen of apple white rot disease. This finding could facilitate better understanding of A. alternata pathogenicity and is relevant for development of biocontrol methods of fungal diseases.

Plants and fungi are closely associated through parasitic or symbiotic relationships in which bidirectional exchanges of cellular contents occur. Recently, a plant virus was shown to be transmitted from a plant to a fungus, but it is unknown whether fungal viruses can also cross host barriers and spread to plants. In this study, we investigated the infectivity of Cryphonectria hypovirus 1 (CHV1, family Hypoviridae), a capsidless, positive-sense (+), single-stranded RNA (ssRNA) fungal virus in a model plant, Nicotiana tabacum. CHV1 replicated in mechanically inoculated leaves but did not spread systemically, but coinoculation with an unrelated plant (+)ssRNA virus, tobacco mosaic virus (TMV, family Virgaviridae), or other plant RNA viruses, enabled CHV1 to systemically infect the plant. Likewise, CHV1 systemically infected transgenic plants expressing the TMV movement protein, and coinfection with TMV further enhanced CHV1 accumulation in these plants. Conversely, CHV1 infection increased TMV accumulation when TMV was introduced into a plant pathogenic fungus, Fusarium graminearum. In the in planta F. graminearum inoculation experiment, we demonstrated that TMV infection of either the plant or the fungus enabled the horizontal transfer of CHV1 from the fungus to the plant, whereas CHV1 infection enhanced fungal acquisition of TMV. Our results demonstrate two-way facilitative interactions between the plant and fungal viruses that promote cross-kingdom virus infections and suggest the presence of plant–fungal-mediated routes for dissemination of fungal and plant viruses in nature.

Viroids are pathogenic agents that have a small, circular noncoding RNA genome. They have been found only in plant species; therefore, their infectivity and pathogenicity in other organisms remain largely unexplored. In this study, we investigate whether plant viroids can replicate and induce symptoms in filamentous fungi. Seven plant viroids representing viroid groups that replicate in either the nucleus or chloroplast of plant cells were inoculated to three plant pathogenic fungi, Cryphonectria parasitica, Valsa mali, and Fusarium graminearum. By transfection of fungal spheroplasts with viroid RNA transcripts, each of the three, hop stunt viroid (HSVd), iresine 1 viroid, and avocado sunblotch viroid, can stably replicate in at least one of those fungi. The viroids are horizontally transmitted through hyphal anastomosis and vertically through conidia. HSVd infection severely debilitates the growth of V. mali but not that of the other two fungi, while in F. graminearum and C. parasitica, with deletion of dicer-like genes, the primary components of the RNA-silencing pathway, HSVd accumulation increases. We further demonstrate that HSVd can be bidirectionally transferred between F. graminearum and plants during infection. The viroids also efficiently infect fungi and induce disease symptoms when the viroid RNAs are exogenously applied to the fungal mycelia. These findings enhance our understanding of viroid replication, host range, and pathogenicity, and of their potential spread to other organisms in nature.plant viroid | fungus | transmission | pathogenicity V iroids are infectious pathogenic agents possessing small, nonencapsidated, circular single-stranded RNAs that, to date, have been found to naturally infect only plants (1, 2). Viroids infect a wide variety of higher plant species, causing devastating diseases in many crops, particularly vegetables, fruits, and ornamental plants (3). In crop plants, viroids are known to spread by vegetative propagation; by mechanical agricultural practices; and, in certain cases, through seeds, pollen, and insect transmission (3, 4). As viroids do not encode any proteins and do not require a helper agent for their multiplication and survival, the biological activities of viroids are entirely dependent on direct interactions of their RNA genome or its derivatives with cellular host components (5-9). Viroid replication or processing of its RNAs in the yeast, Saccharomyces cerevisiae, and cyanobacterium, Nostoc (Nostocales), have been demonstrated (10-12).Currently, at least 34 viroid species have been identified and are classified into two families, Avsunviroidae and Pospiviroidae (13-15). The members of Avsunviroidae (type species: Avocado sunblotch viroid) replicate in the chloroplasts or plastids through symmetric rolling-circle replication using the host nuclear-encoded polymerase. Their RNAs form highly branched secondary structures and have ribozyme activities. Members of Pospiviroidae (type species: Potato spindle tuber viroid) replicate and accumulate in the nucleus th...

MdMYB88 and MdMYB124 have been demonstrated to be responsible for lignin accumulation in apple under drought stress. In this study, using a metabolomic approach, we identified differentially accumulated phenylpropanoid and flavonoid metabolites in MdMYB88/124 transgenic RNAi plants under control and long-term drought stress conditions in apple roots. We confirmed the regulation of phenylalanine by MdMYB88 and MdMYB124 via UPLC-MS in apple roots under both control and drought conditions. Using Electrophoretic Mobility Shift Assay (EMSA) and ChIPquantitative PCR (qPCR) analyses, we found that MdMYB88 positively regulates the MdCM2 gene, which is responsible for phenylalanine biosynthesis, through binding to its promoter region. Under long-term drought conditions, MdMYB88/124 RNAi plants consistently accumulated increased amounts of H 2 O 2 and MDA, while MdMYB88 and MdMYB124 overexpression plants accumulated decreased amounts of H 2 O 2 and MDA. We also examined the accumulation of metabolites in the phenylpropanoid biosynthesis pathway in the leaves of MdMYB88 and MdMYB124 transgenic apple plants after long-term drought stress. We found that metabolites responsible for plant defense, including phenylpropanoids and flavonoids, accumulated less in the RNAi plants but more in the overexpression plants under both control and drought conditions. We further demonstrated that MdMYB88/124 RNAi plants were more sensitive to Alternaria alternata f. sp. mali and Valsa mali, two pathogens that currently severely threaten apple production. In contrast, MdMYB88 and MdMYB124 overexpression plants were more tolerant to these pathogens. The cumulative results of this study provided evidence for secondary metabolite regulation by MdMYB88 and MdMYB124, further explained the molecular roles of MdMYB88 and MdMYB124 in drought resistance, and provided information concerning molecular aspects of their roles in disease resistance.

Viroids are the smallest known infectious agents that are thought to only infect plants. Here, we reveal that several species of plant pathogenic fungi that were isolated from apple trees infected with apple scar skin viroid (ASSVd) carried ASSVd naturally. This finding indicates the spread of viroids to fungi under natural conditions and further suggests the possible existence of mycoviroids in nature. A total of 117 fungal isolates were isolated from ASSVd-infected apple trees, with the majority (85.5%) being an ascomycete Alternaria alternata and the remaining isolates being other plant-pathogenic or -endophytic fungi. Out of the examined samples, viroids were detected in 81 isolates (69.2%) including A. alternata as well as other fungal species. The phenotypic comparison of ASSVd-free specimens developed by single-spore isolation and ASSVd-infected fungal isogenic lines showed that ASSVd affected the growth and pathogenicity of certain fungal species. ASSVd confers hypovirulence on ascomycete Epicoccum nigrum. The mycobiome analysis of apple tree-associated fungi showed that ASSVd infection did not generally affect the diversity and structure of fungal communities but specifically increased the abundance of Alternaria species. Taken together, these data reveal the occurrence of the natural spread of viroids to plants; additionally, as an integral component of the ecosystem, viroids may affect the abundance of certain fungal species in plants. Moreover, this study provides further evidence that viroid infection could induce symptoms in certain filamentous fungi.