Molecular phylogeny and secondary structure analysis of hop stunt viroid (HSVd) associated with Mulberry (Morus alba) in India.

Hop stunt viroid (HSVd), a small, single stranded, circular, non-coding infectious RNA known to cause infection in various economically important crop plants. In the present investigation, a study was conducted in the southern part of Karnataka districts of India to detect the possible association of HSVd infection in mulberry plants. A total of 41 mulberry plants showing typical viroid-like symptoms along with asymptomatic samples were collected and screened using conventional Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) using a specific set of HSVd-Fw/ HSVd-Re primers. Out of 41 samples, the study confirmed the presence of HSVd in six samples of mulberry collected from Ramanagara (1 sample), Chikkaballapur (3 samples) and Doddaballapura (2 samples) regions with an expected HSVd amplicon size of ∼ 290-300 nucleotides. The mechanical transmission of HSVd was also confirmed on cucumber (cv. Suyo) seedlings through bioassay, which was reconfirmed by RT-PCR. The amplicons were cloned, sequenced, and the representative nucleotide sequences were deposited in the NCBI GenBank. Subsequently, molecular phylogenetic analysis showed that HSVd mulberry isolates from this study were most closely related to grapevine isolates, indicating a common origin. On the other hand, it was shown to belong to a different group from mulberry isolates so far reported from Iran, Italy, Lebanon, and China. The secondary structure analysis of HSVd mulberry Indian isolates exhibited substitutions in the terminal left, pathogenicity, and variable regions compared to those of the Indian grapevine isolates. As far as this study is concerned, HSVd was detected exclusively in some mulberry plants with viral-like symptoms, but the pathogenesis and symptom expression needs to be further investigated to establish the relationship between HSVd and the disease symptoms in the mulberry plants.

Precisely Monomeric Linear RNAs of Viroids Belonging to Pospiviroid and Hostuviroid Genera Are Infectious Regardless of Transcription Initiation Site and 5'-Terminal Structure.

Infectious dimeric RNA transcripts are a powerful tool for reverse genetic analyses in viroid studies. However, the construction of dimeric cDNA clones is laborious and time consuming, especially in mutational analyses by in vitro mutagenesis. In this study, we developed a system to synthesize a precisely monomeric linear RNA that could be transcribed in vitro directly from the cDNA clones of four viroid species. The cDNA clones were constructed such that RNA transcription was initiated at the guanine nucleotide of a predicted processing and ligation site in the viroid replication process. Although the transcribed RNAs were considered to possess 5'-triphosphate and 3'-hydroxyl termini, the RNA transcripts were infectious even without in vitro modifications. Additionally, infectivity was detected in the monomeric RNA transcripts, in which transcription was initiated at guanine nucleotides distinct from the predicted processing/ligation site. Moreover, monomeric viroid RNAs bearing 5'-monophosphate, 5'-hydroxyl, or 5'-capped termini were found to be infectious. Northern blot analysis of the pooled total RNA of the plants inoculated with the 5'-terminal modified RNA of potato spindle tuber viroid (PSTVd) indicated that maximum PSTVd accumulation occurred in plants with 5'-monophosphate RNA inoculation, followed by the plants with 5'-triphosphate RNA inoculation. Our system for synthesizing an infectious monomeric linear viroid RNA from a cDNA clone will facilitate mutational analyses by in vitro mutagenesis in viroid research.

Tolerance Even to Lethal Strain of Potato Spindle Tuber Viroid Found in Wild Tomato Species Can Be Introduced by Crossing.

To date, natural resistance or tolerance, which can be introduced into crops by crossing, to potato spindle tuber viroid (PSTVd) has not been reported. Additionally, responses to PSTVd infection in many wild tomato species, including some species that can be crossed with PSTVd-susceptible cultivated tomatoes (Solanum lycopersicum var. lycoperaicum), have not been ascertained. The aim of this study was to evaluate responses to PSTVd infection including resistance and tolerance. Accordingly, we inoculated several cultivated and wild tomato species with intermediate and lethal strains of PSTVd. None of the host plants exhibited sufficient resistance to PSTVd to render systemic infection impossible; however, these plants displayed other responses, including tolerance. Further analysis of PSTVd accumulation revealed low accumulation of PSTVd in two wild species, exhibiting high tolerance, even to the lethal strain. Additionally, F1 hybrids generated by crossing a PSTVd-sensitive wild tomato (Solanum lycopersicum var. cerasiforme) with these wild relatives also exhibited tolerance to the lethal PSTVd strain, which is accompanied by low PSTVd accumulation during early infection. These results indicate that the tolerance toward PSTVd in wild species is a dominant trait and can be utilized for tomato breeding by crossing.

Suppression of RNA-dependent RNA polymerase 6 in tomatoes allows potato spindle tuber viroid to invade basal part but not apical part including pluripotent stem cells of shoot apical meristem.

RNA-dependent RNA polymerase 6 (RDR6) is one of the key factors in plant defense responses and suppresses virus or viroid invasion into shoot apical meristem (SAM) in Nicotiana benthamiana. To evaluate the role of Solanum lycopersicum (Sl) RDR6 upon viroid infection, SlRDR6-suppressed (SlRDR6i) 'Moneymaker' tomatoes were generated by RNA interference and inoculated with intermediate or lethal strain of potato spindle tuber viroid (PSTVd). Suppression of SlRDR6 did not change disease symptoms of both PSTVd strains in 'Moneymaker' tomatoes. Analysis of PSTVd distribution in shoot apices by in situ hybridization revealed that both PSTVd strains similarly invade the basal part but not apical part including pluripotent stem cells of SAM in SlRDR6i plants at a low rate unlike a previous report in N. benthamiana. In addition, unexpectedly, amount of PSTVd accumulation was apparently lower in SlRDR6i plants than in control tomatoes transformed with empty cassette in early infection especially in the lethal strain. Meanwhile, SlRDR6 suppression did not affect the seed transmission rates of PSTVd. These results indicate that RDR6 generally suppresses PSTVd invasion into SAM in plants, while suppression of RDR6 does not necessarily elevate amount of PSTVd accumulation. Additionally, our results suggest that host factors such as RDR1 other than RDR6 may also be involved in the protection of SAM including pluripotent stem cells from PSTVd invasion and effective RNA silencing causing the decrease of PSTVd accumulation during early infection in tomato plants.