Integrative time-scale and multi-omic analysis of host-responses to Hop stunt viroid infection

Constricted by an extreme biological simplicity, viroids are compelled to subvert host regulatory networks in order to accomplish their infectious process. Most of the studies focused on the response to viroid infection have only addressed a specific host regulatory level and considered a unique infection time. Thus, much remains to be done if we want to understand the temporal evolution and complex nature of viroid-host interactions. Here we present an integrative analysis of the timing and intensity of the genome-wide alterations in cucumber plants infected with Hop stunt viroid (HSVd). Differential host transcriptome, sRNAnome and methylome were integrated to determine the temporal response to viroid-infection. Our results support that HSVd promotes a dynamic redesign of the cucumber regulatory pathways predominantly affecting specific regulatory layers at different infection phases. Remarkably, the initial response was characterized by a reconfiguration of the host transcriptome by differential exon usage, followed by a predominant down-regulation of the transcriptional activity possibly modulated by the host epigenetic changes associated to infection and characterized by an increased hypermethylation. The silencing of at least three cucumber transcripts potential targets of vd-sRNAs was also observed. The alteration in host sRNA and miRNA metabolism was marginal. We expect that these data constituting the first comprehensive map of the cucumber-response to HSVd could contribute to elucidate the molecular basis of the host alterations triggered by viroid infection.

La importancia de la aplicación de micorrizas para disminuir la abscisión de naranja (Citrus sinensis L. Osbeck) en Veracruz, México / The importance of the application of mycorrhizae to reduce abscission of orange in Veracruz, Mexico

México es el cuarto productor de cítricos a nivel mundial (FAOSTAT, 2019) y el estado de Veracruz aporta el 53 % de la producción nacional (SIAP, 2019). El problema de la abscisión de frutos de naranja cobró importancia en 2017, pero ya existía este problema desde hace 15 años, lo cual ocasiona pérdidas en la producción del 20% en la zona Norte de Veracruz (Gómez et al., 2020). El ataque de patógenos: Colletotrichum spp, Botryodiplodia spp, Fusarium spp, Citrus tristeza virus (VTC), Candidatus Liberibacter asiaticus (CLas), Citrus exocortis viroid (CEVd) y Hop stunt viroid (HSVd); pueden influir, entre otros factores, en la caída de naranja. El experimento se llevó a cabo en la huerta `Grupo Los Gómez’ en el ejido de San Pablo, Papantla, Veracruz, México; en naranja ‘Valencia’ portainjerto ‘Agrio’ y en suelo tipo aluvión. El objetivo central de la investigación fue evaluar 15 insumos agroecológicos para disminuir la abscisión de naranja, 7 de estos insumos con micorrizas y el resto sin su aplicación. Los tratamientos con mejores resultados fueron: Agua de vidrio (1.25 %) + Micorriza; Optifer amino (0.5 %) + Micorriza; y EM (5 %) + Micorriza con menor porcentaje de caída de naranja con 4.97%, 5.79%, 6.10% respectivamente.

First report of Grapevine yellow speckle viroid 1 infecting grapevine (Vitis vinifera L.) in Canada

Quebec is the third largest wine grape producer in Canada in acreage, tonnage, and wine grape sales (Carisse et al. 2017; Ben Moussa et al. 2019). To evaluate the diversity of viruses infecting grapevine in Quebec, a total of 77 leaf tissue samples (cv. Vidal) were collected from July to October in 2020 in three different vineyards located in Frelighsburg, Hemmingford and Saint-Jacques-le-Mineur in Quebec, Canada. Double-stranded RNA was extracted from each sample and used for cDNA library preparation with the Nextera XT DNA Library Preparation Kit (Illumina) as described previously (Kesanakurti et al. 2016). High-throughput sequencing (HTS, 2x300 bp) was conducted on dual-indexed libraries in a v3 flow cell using the Illumina MiSeq platform (Adkar-Purushothama et al. 2020). The obtained raw FASTQ data was de-multiplexed into 154 separate sequence files, and the adapters and barcode sequences were trimmed. The quality of the sequences was verified using Trimmomatic V.0.32 and the “clean” sequences were analyzed using Virtool and VirFind virus detection pipelines described elsewhere (Ho and Tzanetakis 2014; Rott et al. 2017) to screen for all possible viruses in the databases. Over 100,000 reads per sample were obtained with a percentage of mapped viral reads ranging from 1.47 to 19.43% of total number of reads. Out of 77 samples, 16 revealed the sequence of grapevine yellow speckle viroid 1 (GYSVd-1), for which the length coverage ranged from 98.5 to 99.1%; the depth ranged from 2X to 856X. The GYSVd-1 positive sequence files were subjected to whole genome assembly on CLC genomics Workbench v20.0.4 with the isolate SY-BR from Brazil (KU880715) used as reference. Seven complete genomes of GYSVd-1 of 366-368 nucleotides (nt) in size were deposited (GenBank Acc. MW732682 to MW732688). BLASTN analysis of the sequences showed 98-100% nt identities with isolate SY-BR. Other viruses and viroids such as Grapevine fleck virus, Grapevine rupestris stem pitting-associated virus, Grapevine rupestris vein feathering virus and Hop stunt viroid were also detected. To confirm GYSVd-1 presence in Quebec vineyards, seven of the 16 HTS-positive grapevine leaf tissue samples were subjected to total RNA extraction, followed by RT-PCR assay as before (Adkar-Purushothama et al. 2015; Sahana et al. 2013); all were positive by RT-PCR. The PCR products were directly Sanger-sequenced, and they showed 100% nt identity to the HTS derived sequences. Three of the seven GYSVd-1 positive grapevines exhibited yellow leaf spots and flecks and tiny yellow leaves, but their mixed infection status makes definitive symptoms association difficult to determine. Previously, Hop stunt viroid was reported from grapevines in Canada (Xiao et al. 2019; Fall et al. 2020) but to the best of our knowledge, this is the first report of GYSVd-1 infecting grapevines in Canada, specifically in the province of Quebec. Further research is required to assess the GYSVd-1 related yield loss. Monitoring and testing for GYSVd-1 infection is necessary to prevent propagation of infected materials, spread, and potential negative impact for the Canadian grapevine industry.

Tomato apical stunt viroid.

Tomato apical stunt viroid (TASVd) is a serious pathogen of tomato. Pathways for introduction include tomato seedlings, tomato seeds and ornamentals. If spread to tomato, considerable losses could result. TASVd is spread easily through plant sap, e.g. during pruning and propagation, and there is some evidence of insect transmission in the greenhouse. No symptoms appear on infected ornamental solanaceous plants, but these plants can act as a reservoir for the spread of viroids in tomato production, especially in greenhouse conditions. TASVd outbreaks in tomato are rare although it has occurred in several countries in Asia, Africa and Europe. The economic impact of TASVd in tomato production is not known, but heavy yield losses may result from infection with certain strains. This viroid has not been reported as an invasive species.

Tomato apical stunt viroid.

Tomato apical stunt viroid (TASVd) is a serious pathogen of tomato. Pathways for introduction include tomato seedlings, tomato seeds and ornamentals. If spread to tomato, considerable losses could result. TASVd is spread easily through plant sap, e.g. during pruning and propagation, and there is some evidence of insect transmission in the greenhouse. No symptoms appear on infected ornamental solanaceous plants, but these plants can act as a reservoir for the spread of viroids in tomato production, especially in greenhouse conditions. TASVd outbreaks in tomato are rare although it has occurred in several countries in Asia, Africa and Europe. The economic impact of TASVd in tomato production is not known, but heavy yield losses may result from infection with certain strains. This viroid has not been reported as an invasive species.

Hop stunt viroid (hop stunt viroid).

Hop stunt viroid (HSVd) is a covalently closed, single-stranded RNA molecule of 297 nucleotides (Sano et al., 1985). Variants consisting of 294-303 nucleotides have been described from different hosts (Kofalvi et al., 1997; Amari et al., 2001). HSVd has a rod-like conformation with five domains, a central conserved region (CCR) similar to members of genera Pospiviroid and Cocadviroid, and a terminal conserved hairpin (TCH) which is also present in the genus Cocadviroid (Flores et al., 1997). Replication is known to occur through an asymmetric, rolling-circle model because longer-than-unit minus strands have been found in infected tissue (Flores et al., 2005). HSVd was named due to the first identification of the pathogen on hop plants originating from Japan (Yamamoto et al., 1973). In a wide range of host species, infection by HSVd appears to be latent, whereas in hop, Citrus and Prunus species it causes specific disorders and economic damage (Hadidi et al., 2003).