Viroids, the smallest known pathogens, unable to encode any proteins, can cause severe diseases in their host plants. One of the proposed mechanisms of their pathogenicity includes silencing the ...host's genes via viroid-derived small RNAs, which are products of the host's immune response to the viroid's double stranded RNA. Humulus lupulus (hop) plants are hosts to several viroids; two of them, HLVd and CBCVd, are interesting models for studying host-viroid interactions, due to the symptomless infection of the former and severe stunting disease caused by the latter. To study these interactions, we constructed a deep hop NGS transcriptome based on 35 Gb paired-end sequencing data assembled into over 74 Mb of contigs. These transcripts were used for in-silico prediction of target transcripts of vd-sRNA of the two aforementioned viroids, using two different software tools. Prediction models revealed that 1062 and 1387 hop transcripts share nucleotide similarities with HLVd- and CBCVd-derived small RNAs, respectively, so they could be silenced in an RNA interference process. Furthermore, we selected 17 transcripts from 4 groups of targets involved in the metabolism of plant hormones, small RNA biogenesis, transcripts with high complementarity with viroid-derived small RNAs and transcripts targeted by CBCVd-derived small RNAs with high cellular concentrations. Their expression was monitored by reverse transcription quantitative PCR performed using leaf, flower and cone samples. Additionally, the expression of 5 pathogenesis related genes was monitored. Expression analysis confirmed high expression levels of four pathogenesis related genes in leaves of HLVd and CBCVd infected hop plants. Expression fluctuations were observed for the majority of targets, with possible evidence of downregulation of GATA transcription factor by CBCVd- and of linoleate 13S-lipoxygenase by HLVd-derived small RNAs. These results provide a deep transcriptome of hop and the first insights into complex viroid-hop plant interactions.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Direct crop losses due to plant diseases and the measures used to control them have significant agricultural and economic impacts. The shift from diverse small-scale to large-scale genetically ...uniform monoculture production, along with agricultural intensification and climate change, has led to several known epidemics in man-made agroecosystems that have been rendered more vulnerable to pathogens. One such example is hop growing, which is threatened by highly aggressive hop viroids. Since 2007, almost one-third (about 500 ha) of Slovenian hop gardens have been affected by severe hop stunt disease caused by citrus bark cracking viroid (CBCVd), which continues to spread despite strict prevention measures. We have developed and validated a multiplex RT-qPCR (mRT-qPCR) for the sensitive detection of CBCVd, hop latent viroid (HLVd), and hop stunt viroid (HSVd). Singleplex RT-qPCR assays were designed individually and subsequently combined in a one-step mRT-qPCR assay. Hop-specific mRNA170 and mRNA1192 internal controls were also developed to detect possible PCR inhibition. Analytical specificity was tested on 35 samples from different hosts, geographic regions, and combinations of viroids. Method validation showed that mRT-qPCR had lower sensitivity than singleplex RT-qPCR, while specificity, selectivity, repeatability, and reproducibility remained unchanged. The newly developed assays were found to be robust, reliable, and suitable for large-scale screening of hop viroids.
During fungal infections, plant cells secrete chitinases, which digest chitin in the fungal cell walls. The recognition of released chitin oligomers via lysin motif (LysM)-containing immune host ...receptors results in the activation of defense signaling pathways. We report here that Verticillium nonalfalfae, a hemibiotrophic xylem-invading fungus, prevents these digestion and recognition processes by secreting a carbohydrate-binding motif 18 (CBM18)-chitin-binding protein, VnaChtBP, which is transcriptionally activated specifically during the parasitic life stages. VnaChtBP is encoded by the Vna8.213 gene, which is highly conserved within the species, suggesting high evolutionary stability and importance for the fungal lifestyle. In a pathogenicity assay, however, Vna8.213 knockout mutants exhibited wilting symptoms similar to the wild-type fungus, suggesting that Vna8.213 activity is functionally redundant during fungal infection of hop. In a binding assay, recombinant VnaChtBP bound chitin and chitin oligomers in vitro with submicromolar affinity and protected fungal hyphae from degradation by plant chitinases. Moreover, the chitin-triggered production of reactive oxygen species from hop suspension cells was abolished in the presence of VnaChtBP, indicating that VnaChtBP also acts as a suppressor of chitin-triggered immunity. Using a yeast-two-hybrid assay, circular dichroism, homology modeling, and molecular docking, we demonstrated that VnaChtBP forms dimers in the absence of ligands and that this interaction is stabilized by the binding of chitin hexamers with a similar preference in the two binding sites. Our data suggest that, in addition to chitin-binding LysM (CBM50) and Avr4 (CBM14) fungal effectors, structurally unrelated CBM18 effectors have convergently evolved to prevent hydrolysis of the fungal cell wall against plant chitinases and to interfere with chitin-triggered host immunity.
Viroids are small non-capsidated, single-stranded, covalently-closed circular noncoding RNA replicons of 239-401 nucleotides that exploit host factors for their replication, and some cause disease in ...several economically important crop plants, while others appear to be benign. The proposed mechanisms of viroid pathogenesis include direct interaction of the genomic viroid RNA with host factors and post-transcriptional or transcriptional gene silencing via viroid-derived small RNAs (vd-sRNAs) generated by the host defensive machinery.
(hop) plants are hosts to several viroids among which
(HLVd) and
(CBCVd) are attractive model systems for the study of viroid-host interactions due to the symptomless infection of the former and severe symptoms induced by the latter in this indicator host. To better understand their interactions with hop plant, a comparative transcriptomic analysis based on RNA sequencing (RNA-seq) was performed to reveal the transcriptional alterations induced as a result of single HLVd and CBCVd infection in hop. Additionally, the effect of HLVd on the aggressiveness of CBCVd that underlies severe stunting in hop in a mixed infection was studied by transcriptomic analysis. Our analysis revealed that CBCVd infection resulted in dynamic changes in the activity of genes as compared to single HLVd infection and their mixed infection. The differentially expressed genes that are involved in defense, phytohormone signaling, photosynthesis and chloroplasts, RNA regulation, processing and binding; protein metabolism and modification; and other mechanisms were more modulated in the CBCVd infection of hop. Nevertheless, Gene Ontology (GO) classification and pathway enrichment analysis showed that the expression of genes involved in the proteolysis mechanism is more active in a mixed infection as compared to a single one, suggesting co-infecting viroids may result in interference with host factors more prominently. Collectively, our results provide a deep transcriptome of hop and insight into complex single HLVd, CBCVd, and their coinfection in hop-plant interactions.
RNA interference is an evolutionary conserved mechanism by which organisms regulate the expression of genes in a sequence-specific manner to modulate defense responses against various abiotic or ...biotic stresses. Hops are grown for their use in brewing and, in recent years, for the pharmaceutical industry. Hop production is threatened by many phytopathogens, of which
, the causal agent of Verticillium wilt, is a major contributor to yield losses. In the present study, we performed identification, characterization, phylogenetic, and expression analyses of three Argonaute, two Dicer-like, and two RNA-dependent RNA polymerase genes in the susceptible hop cultivar Celeia and the resistant cultivar Wye Target after infection with
. Phylogeny results showed clustering of hop RNAi proteins with their orthologues from the closely related species
,
and
which form a common cluster with species of the Rosaceae family. Expression analysis revealed downregulation of argonaute 2 in both cultivars on the third day post-inoculation, which may result in reduced AGO2-siRNA-mediated posttranscriptional gene silencing. Both cultivars may also repress ta-siRNA biogenesis at different dpi, as we observed downregulation of argonaute 7 in the susceptible cultivar on day 1 and downregulation of RDR6 in the resistant cultivar on day 3 after inoculation.
The vascular plant pathogen Verticillium nonalfalfae causes Verticillium wilt in several important crops. VnaSSP4.2 was recently discovered as a V. nonalfalfae virulence effector protein in the xylem ...sap of infected hop. Here, we expanded our search for candidate secreted effector proteins (CSEPs) in the V. nonalfalfae predicted secretome using a bioinformatic pipeline built on V. nonalfalfae genome data, RNA-Seq and proteomic studies of the interaction with hop. The secretome, rich in carbohydrate active enzymes, proteases, redox proteins and proteins involved in secondary metabolism, cellular processing and signaling, includes 263 CSEPs. Several homologs of known fungal effectors (LysM, NLPs, Hce2, Cerato-platanins, Cyanovirin-N lectins, hydrophobins and CFEM domain containing proteins) and avirulence determinants in the PHI database (Avr-Pita1 and MgSM1) were found. The majority of CSEPs were non-annotated and were narrowed down to 44 top priority candidates based on their likelihood of being effectors. These were examined by spatio-temporal gene expression profiling of infected hop. Among the highest in planta expressed CSEPs, five deletion mutants were tested in pathogenicity assays. A deletion mutant of VnaUn.279, a lethal pathotype specific gene with sequence similarity to SAM-dependent methyltransferase (LaeA), had lower infectivity and showed highly reduced virulence, but no changes in morphology, fungal growth or conidiation were observed. Several putative secreted effector proteins that probably contribute to V. nonalfalfae colonization of hop were identified in this study. Among them, LaeA gene homolog was found to act as a potential novel virulence effector of V. nonalfalfae. The combined results will serve for future characterization of V. nonalfalfae effectors, which will advance our understanding of Verticillium wilt disease.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Hop cultivation, integral to the brewing industry, faces challenges from viroids, especially the
citrus bark cracking viroid
(CBCVd) but also the
hop latent viroid
(HLVd) influences hop cone quality. ...We focused on the degradation kinetics of HLVd thereby covering compost, silage, and digestate made from hop residues. In this study, HLVd serves as a model for understanding CBCVd, which causes significant stunting and yield losses in European hop crops. Composting experiments revealed that although composting significantly lowers HLVd levels, complete degradation within 7 weeks is not guaranteed, with loose compost showing a more rapid reduction than compacted variants. Infectivity experiments conducted using inocula obtained from HLVd-infected hop plant residues exposed to composting, ensiling, and biogas digestate did not result in the transmission of HLVd to viroid-free plants. Also extracting and analyzing the soil-root mixture of plants inoculated with HLVd-infected hop residues did not show evidence for viroid persistence. Degradation experiments further differentiated between the physiochemical and biological influences on viroid and viroid-like random RNA stability, showing that higher temperatures of 50 °C enhance degradation over 40 °C, and pH levels of 5 or 7 are slowing degradation. In contrast deionized water or a pH of 4 or 9 enhances viroid degradation. Adding extracts from digestate accelerated the process indicating a role of biological activity. Interestingly, a viroid-like random RNA with similar physiochemical properties, showed to degrade faster compared to HLVd, suggesting high robustness of the actual viroid secondary structure. These findings offer valuable insights into managing HLVd in hops and potentially other crops, highlighting effective strategies to mitigate viroid spread, and contributing to broader understanding of RNA degradation in agriculture.
(
) is one of the most problematic hop (
L.) pathogens, as the highly virulent fungal pathotypes cause severe annual yield losses due to infections of entire hop fields. In recent years, the RNA ...interference (RNAi) mechanism has become one of the main areas of focus in plant-fungal pathogen interaction studies and has been implicated as one of the major contributors to fungal pathogenicity. MicroRNA-like RNAs (milRNAs) have been identified in several important plant pathogenic fungi; however, to date, no milRNA has been reported in the
species. In the present study, using a high-throughput sequencing approach and extensive bioinformatics analysis, a total of 156 milRNA precursors were identified in the annotated
genome, and 27 of these milRNA precursors were selected as true milRNA candidates, with appropriate microRNA hairpin secondary structures. The stem-loop RT-qPCR assay was used for milRNA validation; a total of nine
milRNAs were detected, and their expression was confirmed. The milRNA expression patterns, determined by the absolute quantification approach, imply that milRNAs play an important role in the pathogenicity of highly virulent
pathotypes. Computational analysis predicted milRNA targets in the
genome and in the host hop transcriptome, and the activity of milRNA-mediated RNAi target cleavage was subsequently confirmed for two selected endogenous fungal target gene models using the 5' RLM-RACE approach.
The conserved RNA interference mechanism (RNAi) in the fungal kingdom has become a focus of intense scientific investigation. The three catalytic core components, Dicer-like (DCL), Argonaute (AGO), ...and RNA-dependent RNA polymerase (RdRP), and their associated small interfering RNA molecules (siRNAs) have been identified and characterised in several fungal species. Recent studies have proposed that RNAi is a major contributor to the virulence of fungal pathogens as a result of so-called trans-kingdom RNA silencing. In the present study, we report on the existence of three core RNAi proteins in the pathogenic plant fungus Verticillium nonalfalfae, which is a soilborne plant pathogen that causes severe wilting disease in hops (Humulus lupulus L.). Two DCL proteins, two AGO proteins, and two RdRP proteins were identified, and their conserved RNAi domains were characterised. Our phylogeny results confirm the existing taxonomic relationships in the Ascomycete fungal phylum and show that the fungi of the Hypocreomycetidae subclass of the Sordariomycetes class have high amino acid sequence similarity. The expression analysis revealed a potential role of RNAi in the pathogenicity of the fungi, since all the RNAi genes were highly upregulated in the highly virulent isolate T2 and were also differentially expressed in the V. nonalfalfae-susceptible Celeia and V. nonalfalfae-resistant Wye Target cultivars.
Verticillium dahliae
Kleb., the causal agent of Verticillium wilts, is a devastating plant disease affecting many plant species. Fungus
V. dahliae
was detected in a partially artificially established
...Acer pseudoplatanus
L. forest stand in central Slovenia. This finding incited further study about the risk of different sources of
V. dahliae
isolates for maples in forests and the pathogenicity of three
V. dahliae
isolates of different origins was tested on saplings of
A. pseudoplatanus
,
A. platanoides
L., and
A. campestre
L. The inoculated saplings exhibited disease symptoms, i.e., leaf necrosis and wilting. At the end of the pathogenicity test, typical xylem browning was visible on the cross-sections, and the pathogen was successfully re-isolated. The isolates showed significant differences in their pathogenicity to specific maple hosts, with the agricultural isolate (originated from bell pepper) being the most aggressive on all three maple species. The disease severity index (DSI) and relative area under the disease progress curve (rAUDPC), as well as the success of re-isolation, indicate that
A. platanoides
is the most susceptible to inoculation with
V. dahliae
. In addition, significant differences in sapling biomass were observed between treated and control plants. These results suggest that maples in forest stands are threatened by
V. dahliae
, and biosecurity measures should be considered and implemented in forest management to reduce the transmission and potential spread of the pathogen.
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