RNA‐directed DNA methylation efficiency depends on trigger and target sequence identity

RNA‐directed DNA methylation (RdDM) in plants has been extensively studied, but the RNA molecules guiding the RdDM machinery to their targets are still to be characterized. It is unclear whether these molecules require full complementarity with their target. In this study, we have generated Nicotiana tabacum (Nt) plants carrying an infectious tomato apical stunt viroid (TASVd) transgene (Nt‐TASVd) and a non‐infectious potato spindle tuber viroid (PSTVd) transgene (Nt‐SB2). The two viroid sequences exhibit 81% sequence identity. Nt‐TASVd and Nt‐SB2 plants were genetically crossed. In the progeny plants (Nt‐SB2/TASVd), deep sequencing of small RNAs (sRNAs) showed that TASVd infection was associated with the accumulation of abundant small interfering RNAs (siRNAs) that mapped along the entire TASVd but only partially matched the SB2 transgene. TASVd siRNAs efficiently targeted SB2 RNA for degradation, but no transitivity was detectable. Bisulfite sequencing in the Nt‐SB2/TASVd plants revealed that the TASVd transgene was targeted for dense cis‐RdDM along its entire sequence. In the same plants, the SB2 transgene was targeted for trans‐RdDM. The SB2 methylation pattern, however, was weak and heterogeneous, pointing to a positive correlation between trigger–target sequence identity and RdDM efficiency. Importantly, trans‐RdDM on SB2 was also detected at sites where no homologous siRNAs were detected. Our data indicate that RdDM efficiency depends on the trigger–target sequence identity, and is not restricted to siRNA occupancy. These findings support recent data suggesting that RNAs with sizes longer than 24 nt (>24‐nt RNAs) trigger RdDM.     

Influence of virus and virus-like agents on the development of citrus buds cultured in vitro

Tissue culture in vitro was used to determine the effect of six major citrus virus and virus-like agents. Nodal stem segments from inoculated Pineapple sweet orange (Citrus sinensis (L.) Osb.), Mexican lime (C. aurantifolia (Christm.) Swing.) and Arizona Etrog citron 861-Sl (C. medica L.) were cultured in vitro to induce shoots. Some virus and virus-like agents had a marked effect on bud development and further recovery of plantlets. The number and size of the shoots that developed from each bud were affected as a result of infection. The effect depended on the specific virus, the isolate and the host-disease combination. The possible implications of these results are discussed.     

Simultaneous Detection of Three Viroid Species Infecting Hops in China by Multiplex RT‐PCR

We have developed a multiplex RT‐PCR protocol for the simultaneous detection of three viroids in three different genera that infect hops: Hop latent viroid (HLVd; Cocadviroid), Hop stunt viroid (HSVd; Hostuviroid) and Apple fruit crinkle viroid (AFCVd; Apscaviroid). The method was validated by testing 175 hop samples collected from the Xinjiang autonomous region of China. All samples were found to be positive for HLVd but negative for AFCVd, confirming the widespread or even ubiquitous infection of HLVd and the low incidence of AFCVd in hops in China. In addition, HSVd was detected in 22.86% of the samples tested. This rapid and reliable multiplex RT‐PCR assay provides an effective method for detection of three important viroid species in large‐scale surveys for disease management in hops.     

In silico predicted robustness of viroid RNA secondary structures. II. Interaction between mutation pairs

Viroids are plant subviral pathogens whose genomes are constituted by a single-stranded and covalently closed small RNA molecule that does not encode for any protein. Most of the 29 described viroid species fold into a rodlike or quasi-rodlike structure, whereas a few of them fold as highly branched structures. In a previous study, we used RNA thermodynamic secondary structure prediction algorithms to compare the mutational robustness of all viroid species. Here we used the same approach to explore the sign and strength of epistasis among pairs of random mutations. We found that antagonistic interactions were more abundant than synergistic ones. However, despite their lower frequency, synergistic interactions tended to be more intense. Mutational robustness and the intensity of epistasis were correlated such that viroid species with large average mutational effects showed stronger antagonistic epistasis, whereas viroids with mild average mutational effects showed weaker antagonistic interactions. The strength of antagonistic epistasis decreased with genome complexity as a consequence of the gained robustness of duplicated genomes. In good agreement with our previous finding of an evolutionary trend toward increased robustness, we now found a trend toward reduced antagonistic epistasis.     

Comparison of multimeric plus and minus forms of viroids and virusoids

Summary In order to investigate the mechanism of replication of viroids and virusoids, we have compared the replication intermediates of three members of each group in nucleic acid extracts of infected plants. Viroids were avocado sunblotch viroid (ASBV), citrus exocortis viroid (CEV) and coconut cadang cadang viroid (CCCV). Virusoids were from velvet tobacco mottle virus (VTMoV), solanum nodiflorum mottle virus (SNMV) and lucerne transient streak virus (LTSV). Analysis of intermediates was by the Northern hybridization technique with single-strand DNA and RNA probes prepared from recombinant DNA clones. The results obtained are discussed in terms of current models of viroid and virusoid replication. The plus RNA species consisted of an oligomeric series up to decamers based on the unit of full-length viroid or virusoid, which was always the major component, except for CEV where only monomer and dimer species were found. In the case of ASBV and the virusoids of VTMoV and SNMV, a minor, multimeric series of components (X-bands) was superimposed on the main oligomeric series. The complementary minus species proved more difficult to detect and characterise, with each viroid and virusoid exhibiting a unique pattern on Northern hybridization. However, they all had greater than unit-length minus species. In addition, minus species analogous to the plus X-bands were found in ASBV and CEV. The experimental difficulties encountered in this work are discussed in terms of the problem of detecting minus species by Northern analysis in the presence of excess complementary plus species.