RESUMO
Blackcurrant reversion virus (BRV) is the most destructive mite-transmitted pathogen in blackcurrants. The understanding of the resistance to BRV is limited, hindering and delaying the selection process. To identify the resistance (R) gene for BRV resistance, a gene expression analysis based on de novo blackcurrant cv. Aldoniai comparative transcriptome analysis (mock- and BRV-inoculated samples at 2 and 4 days post-inoculation (dpi)) was performed. In this study, 111 annotated clusters associated with pathogenesis according to conservative R gene domains were identified. In virus-infected samples, only Cluster-12591.33361 showed significant expression at 4 dpi. The expression profiles of this cluster were significantly associated with the presence of BRV particles in plant tissues, making it a putative R gene in the dominant resistance strategy in the BRV-Ribes nigrum interaction. The newly identified gene R.nigrum_R belongs to the CC-NBS-LRR class and has 63.9% identity with RPM1 in Populus spp. This study provides new insights on dominant putative R genes related to resistance to BRV in R. nigrum, which could aid targeted research and genetic improvement in breeding programs of blackcurrants.
RESUMO
The most damaging pathogen in blackcurrant plantations is mite-transmitted blackcurrant reversion virus (BRV). Some Ribes species have an encoded genetic resistance to BRV. We performed RNA sequencing analysis of BRV-resistant blackcurrant cv. Aldoniai to evaluate the molecular mechanisms related to the BRV infection response. The RNA of virus-inoculated and mock-inoculated microshoots was sequenced, and the transcriptional changes at 2- and 4-days post inoculation (dpi) were analyzed. The accumulation and expression of BRV RNA1 were detected in infected plants. In total, 159,701 transcripts were obtained and 30.7% were unigenes, annotated in 7 databases. More than 25,000 differentially expressed genes (DEGs) according to FPKM were upregulated or downregulated. We observed 221 and 850 DEGs at 2 and 4 dpi, respectively, in BRV-infected microshoots related to the stress response. The proportion of upregulated DEGs at 4 dpi was about 3.5 times higher than at 2 dpi. Pathways of the virus defense response were activated, and key candidate genes were identified. The phenylpropanoid and the cutin, suberine, and wax biosynthesis pathways were activated in infected plants. Our comparative de novo analysis of the R. nigrum transcriptome provides clues not only for understanding the molecular BRV resistance mechanisms but also for breeding BRV-tolerant genotypes.
Assuntos
Doenças das Plantas , Viroses , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Melhoramento Vegetal , Doenças das Plantas/genética , TranscriptomaRESUMO
Blackcurrant reversion virus (BRV) is the most destructive currant-infecting and mite-transmitted pathogen from the genus Nepovirus. In this work, BRV transmission in the system Ribes ex vitro-Ribes in vitro was applied for the first time. Triple infection of BRV identified in blackcurrant cv. Gojai was used for phylogenetic analysis and inoculation assay. Transmission of BRV was successful due to its stability in the inoculum for up to 8 days at 4 °C; all BRV isolates were infectious. Our suggested inoculation method through roots was applied in six Ribes spp. genotypes with 100.0% reliability, and the expression levels of defence-related gene PR1 to biotic stress was observed. The prevalence of the virus in microshoots after 2-14 days post-inoculation (dpi) was established by PCR. In resistant genotypes, the BRV was identified up to 8 dpi; meanwhile, infection remained constant in susceptible genotypes. We established that BRV transmission under controlled conditions depends on the inoculum quality, post-inoculation cultivation temperature, and host-plant susceptibility to pathogen. This in vitro inoculation method opens possibilities to reveal the resistance mechanisms or response pathways to BRV and can be used for the selection of resistant Ribes spp. in breeding programs.
RESUMO
In response to pathogen attacks, plants activate a complex of defense mechanisms including an accumulation of the endogenous signaling compounds salicylic acid and jasmonic acid. The activity of pathogenesis-related genes (PRs) and coronatine-insensitive 1 (COI1) in defense-response pathways are established in plants. The aim of this study was to identify homologs of the PRs and COI1 in blackcurrants. Primers with degenerate nucleotides were designed based on the most conservative parts of PR1 and COI1 genes from other plants and applied for amplification of specific fragments of PRs and COI1 in Ribes spp. Seven heterogeneous sequences of PR with a diversity of 66.0-98.3% at nucleic acid level were found. The phylogenetic analysis revealed the dependence of R. nigrum PR homologs on the PR1 and PR6 families. Four heterogeneous sequences of R. nigrum COI1 with an identity of 95.9-98.8% at nucleic acid level were isolated. Specific primers for newly detected genes' homologs were designed in this study and could be useful for evaluating the defense response to pathogen attacks in blackcurrants.
