Symptomatic plant viroid infections in phytopathogenic fungi

Abstract

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...

A Single ssRNA Segment Encoding RdRp Is Sufficient for Replication, Infection, and Transmission of Ourmia-Like Virus in Fungi

“…Nerva et al (2017) presented strong evidence to show that a virus that infects an endophytic fungus could replicate in plants. Additionally, viroids have been shown to infect fungi efficiently (Wei et al, 2019). These findings further confirmed that some mycoviruses are close relatives to plant viruses.…”

Symptomatic plant viroid infections in phytopathogenic fungi: A request for a critical reassessment

“…1A). Conversely, the inocula used before (3) were monomeric RNAs with arbitrary termini flanked by vector sequences that may reduce/ abolish their infectivity (8,9) and that were not bioassayed in diagnostic host plants (3). In a second experiment, blocks of N. benthamiana were inoculated, instead of mechanically, by agroinfiltration (far more efficient) with Agrobacterium tumefaciens harboring plasmids for expressing dimeric head-to-tail transcripts of PSTVd, PLMVd, and ASBVd.…”

Reply to Serra et al.: Nucleotide substitutions in plant viroid genomes that multiply in phytopathogenic fungi

“…This is our response to the letter by Serra et al (1), which questioned our recent paper (2) describing plant viroid infections in phytopathogenic fungi. In this study, full-length monomeric cDNA clones of seven plant viroid RNA genomes were produced using oligonucleotide synthesis (2). We opted for the monomeric version of viroid cDNA clone, considering that it requires shorter sequences for oligonucleotide synthesis and because of noting an article showing the infectivity of the monomeric cDNA clone of a viroid (3).…”

“…We opted for the monomeric version of viroid cDNA clone, considering that it requires shorter sequences for oligonucleotide synthesis and because of noting an article showing the infectivity of the monomeric cDNA clone of a viroid (3). We constructed plasmid constructs for in vitro transcriptions by placing the viroid cDNA sequence immediately after a T7 promoter sequence and incorporating a restriction site (HindIII or SpeI) into the 3′ terminus (2). Due to the large number of samples, the infectivity of monomeric viroid cDNA transcripts was assayed by RT-PCR, and indeed, all of the RT-PCR products derived from the detection of viroid RNA accumulation in plants, yeast, and fungi inoculated with viroid RNA transcripts were verified by DNA sequencing.…”

“…After screening for 21 viroid-fungal host combinations, we obtained six combinations that showed stable viroid accumulation in the fungi (2). For each inoculation experiment, a viroid-free fungus was also cultured in parallel and subjected to RT-PCR analysis.…”