Viroids belong to a group of economicaly important plant pathogens on hop, since they are inducers of several uncurable diasesase which threatened production. Some viroid diseases are complex of multiple infections of several viroids where role of individual viroid is not defined yet. In our study we analysed interactions of 4 hop viroids: Citrus bark cracking viroid (CBCVd), Apple fruit crinkle viroid (AFCVd), Hop latent viroid (HLVd), and Hop stunt viroid (HSVd). The analysis revealed low CBCVd compatibility with HSVd, while all other viroid combinations were highly compatible. Unlike to other viroids, single CBCVd propagation showed a significant excess of (−) over (+) strands in hop, tomato, and Nicotiana benthamiana, but not in citruses. Inoculation of hop with all viroids led to multiple infections with unstable viroid levels in individual plants in the pre- and post-dormancy periods, and to high plant mortality and morphological disorders. CBCVd and AFCVd caused a suppression of some crucial mRNAs related to the hop prenylflavonoid biosynthesis pathway, and disbalance of WRKY/WDR1 complexes important for activation of lupulin genes.
COBISS.SI-ID: 8709241
Citrus bark cracking viroid (CBCVd) is recently discovered pathogen of hop, which has been causing severe hop stunt disease. Because of high level of aggressiveness it is interesting for studies od pathogenesis which is in viroid disease still not vell understood. To identify miRNAs in hop and their response to CBCVd-infection, two small RNA (sRNA) libraries prepared from healthy and CBCVd-infected hop plants and were investigated by high throughput sequencing. A total of 36 conserved and 37 novel miRNAs were found to be differentially recovered in the response to CBCVd-infection. The majority of predicted targets belonged to transcriptional factors that may regulate hop leaf, root and cone growth and development. Moreover, the identified miRNAs may also be involved in other cellular and metabolic processes, such as stress response, signal transduction, and other physiological processes including prenylflavonoid biosynthesis pathways. Quantitative real time PCR analysis of selected targets revealed their negative correlation with their corresponding CBCVd-responsive miRNAs. This study present one of the first insight to viroid pathogenesis and have importance in future hop breeding process.
COBISS.SI-ID: 8522361
Hop is a host of viroids CBCVd and HLVd which are inducers of aggressive and nonaggressive symptoms respectively, what it is an interesting model for studying viroid targets responsible for disease symptoms. With the aim to study viroid pathogenicity based on gene silencing we develop hop transcriptome by using NGS (next generation sequencing) technology. To analyse the interaction between vd-sRNA and hop transcriptkon we first assembled hop transcriptome and in silico predicted all possible vd-sRNAs (21, 22 and 24 bp) of CBCVd and HLVd. Prediction models revealed that 1062 and 1387 hop transcripts share nucleotide homologies with HLVd- and CBCVd-derived small RNAs, respectively, so they could be silenced in an RNA interference process. For further work we made target selection based on following categories 1) targets participte in metabolism of plant hormones, 2) targets participate in small RNA biogenesis, 3) CBCVd targets of vd-sRNAs from extremely well covered regions of the viroid, and 4) Genes with high homology with vd-sRNAs. Changes of target expression were checked by reverse transcription coupled with quantitative PCR (RT-qPCR). Elected targets shown changed patterns of expression in the various tissues of the hops. For LOX and GATA transcripts we found evidence of a possible reduced expression. These results provide a deep transcriptome of hop and the first insights into complex viroid-hop plant interactions.
COBISS.SI-ID: 8793977
Hop latent viroid (HLVd) in most hop varieties does not produce visible symptoms; however, it has a negative impact on both the quantity and quality of the crop. The unexpressed symptoms of the infection and its spreading by vegetative propagation are the reason, that it has been present in the majority of hop production regions. In the case of viroid diseases, the use of plant protection products is not possible, so the prevention of spread of the disease is based on sanitary measures and production of healthy planting material. In this case, reliable and rapid method for detection of viroids, are crucial. For identification of HLVd in hops different methods such are polyacrylamide electrophoresis (PAGE) and hybridization are used, but the most commonly used is reverse transcription combined with polymerase chain reaction (RT-PCR). In order to upgrade the existing methods for HLVd detection, we have developed real time RT-PCR (RT-qPCR), which is distinguished by a high degree of sensitivity, speed, analyst-friendly handling and the possibility of upgrading to multiplex detection of other pathogens that are also detected in hop.
COBISS.SI-ID: 8596089
Viroids are the smallest plant pathogens, consisting only of a circular RNA molecule, between 246 and 401 nucleotides in size. Despite the simple structure, they can cause very serious diseases on a wide range of hosts, among which there are many economically important agricultural plants, ornamental plants and even some tree species. Not even hops is an exception, which can be infected by hop latent viroid (HLVd), hop stunt viroid (HSVd), citrus bark cracking viroid (CBCVd) and apple fruit crinkle viroid (AFCVd). The CBCVd viroid is currently present on hops only in Slovenia, causing severe hop stunt disease. The disease is very aggressive, because it causes the degradation and dying of plants with a rapid dynamic of spread. CBCVd is always present in hops in combination with HLVd, which makes etiological study of the impact of CBCVd on hop plants difficult. A method of artificial inoculation with CBCVd was therefore developed, in monomer and dimer form. The designed monomer and dimer were inserted into the plasmid, which served as the basis for RNA transcription. Viroid RNA was used for artificial infection of hops, tomatoes and cucumbers. Each of the host plants was infected with both the monomeric and dimeric CBCVd, as well as total nucleic acids isolated from the infected hop. The CBCVd dimer showed a high degree of infectivity and thus suitability for performing artificial infections.
COBISS.SI-ID: 8906361