Development of hop transcriptome to support research into host-viroid interactions

Abstract

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.

Progress Towards Plant Community Transcriptomics: Pilot RNA-Seq Data from 24 Species of Vascular Plants at Harvard Forest

“…The assemblies are also reasonably complete with more than 70% of BUSCO genes present on average. This is a similar distribution of BUSCO scores to those in the recently published 1KP project One Thousand Plant Transcriptomes Initiative, 2019) and other studies (Blande et al, 2017;Evkaikina et al, 2017;Pokorn et al, 2017;Weisberg et al, 2017) . Like many transcriptome assemblies (Johnson et al, 2012;Carpenter et al, 2019;Patterson et al, 2019) , these assemblies also contain a large number of small scaffolds (<300 bp).…”

Dissection of Dynamic Transcriptome Landscape of Leaf, Bract, and Lupulin Gland in Hop (Humulus lupulus L.)

“…The length-frequency distribution ( Figure 3A), N50, N75 value of de novo assembly, average GC content, and the average lengths of unigenes (Table 1), represents the broad view of all three unigene sets and combined transcriptomes. The comparison analysis showed that the N50 and the average length of unigenes derived from three libraries were much longer than those of any specific tissue and previous studies on hop (N50: 650 bp) [32], suggesting the high quality of sequencing data. Pearson's correlation coefficient between biological replicates were approximately 0.9, suggesting the high reproducibility across samples and credibility of RNA-seq results (Supplementary Materials Figure S1).…”

“…In the present study, a total of 128 million clean reads were obtained after trimming and performing error corrections from the three distinct tissues, representing 12.94 Gb sequences were de novo assembled into a total of 43,550 unigenes ( Table 1). The number of transcripts and an average N50 length of assembled data was slightly higher than RNA-Seq reports of hop genome [32], indicating that sequencing data including transcriptomes were of high quality and integrity and could serve as a basis for functional gene studies and marker development in hop.…”

Studying strands polarity of different viroids and their combinations in infected hop plants

“…Zanj smo uporabili programski paket R (paket Rcmdr, različica 2.4-x). (Rahman et al, 2008;Pokorn et al, 2017).…”