The titre of the virus in the inoculum affects the titre of the viral RNA in the host plant and the occurrence of the disease symptoms

Potato virus Y (PVY) is the most economically important potato virus, therefore extensive research is focusing on elucidation of its interaction with the host. To obtain repeatable results, strict standardization of research methods is crucial. Mechanical inoculation by rubbing sap from a PVY infected plant onto the leaf surface together with a fine abrasive powder is the most convenient way of experimental transmission of PVY to host plants. However, factors determining reproducibility of this process need to be determined. In the present study, it was shown that higher titre of the virus in the inoculum resulted in faster increase of PVYNTN RNA titre in the inoculated leaves, as well as in faster translocation of PVYNTN from inoculated leaves into upper non-inoculated leaves. The final titre of PVYNTN RNA in upper non-inoculated leaves was independent of the virus titre in the inoculum. In addition, the occurrence of the disease symptoms was followed and the dependence to the titre of the virus in the inoculum was observed

Multiomics analysis of tolerant interaction of potato with potato virus Y

Potato virus Y (PVY) is the most economically important viral pathogen of potato worldwide. Different potato cultivars react to the pathogen differently, resulting in resistant, tolerant or disease outcome of the interaction. Here we focus on tolerant interaction between potato cv. Désirée and PVYNTN. To capture the response in its full complexity, we analyzed the dynamic changes on multiple molecular levels, including transcriptomics, sRNAomics, degradomics, proteomics and hormonomics. The analysis was complemented by the measurements of viral accumulation, photosynthetic activity and phenotypisation of the symptoms. Besides cv. Désirée we also studied its transgenic counterpart depleted for the accumulation of salicylic acid (NahG-Désirée). This multiomics analysis provides better insights into the mechanisms leading to tolerant response of potato to viral infection and can be used as a base in further studies of plant immunity regulation

Salicylic Acid Perturbs sRNA-Gibberellin Regulatory Network in Immune Response of Potato to Potato virus Y Infection

Potato virus Y is the most economically important potato viral pathogen. We aimed at unraveling the roles of small RNAs (sRNAs) in the complex immune signaling network controlling the establishment of tolerant response of potato cv. Désirée to the virus. We constructed a sRNA regulatory network connecting sRNAs and their targets to link sRNA level responses to physiological processes. We discovered an interesting novel sRNAs-gibberellin regulatory circuit being activated as early as 3 days post inoculation (dpi) before viral multiplication can be detected. Two endogenous sRNAs, miR167 and phasiRNA931 were predicted to regulate gibberellin biosynthesis genes GA20-oxidase and GA3-oxidase. The increased expression of phasiRNA931 was also reflected in decreased levels of GA3-oxidase transcripts. Moreover, decreased concentration of gibberellin confirmed this regulation. The functional relation between lower activity of gibberellin signaling and reduced disease severity was previously confirmed in Arabidopsis-virus interaction using knockout mutants. We further showed that this regulation is salicylic acid-dependent as the response of sRNA network was attenuated in salicylic acid-depleted transgenic counterpart NahG-Désirée expressing severe disease symptoms. Besides downregulation of gibberellin signaling, regulation of immune receptor transcripts by miR6022 as well as upregulation of miR164, miR167, miR169, miR171, miR319, miR390, and miR393 in tolerant Désirée, revealed striking similarities to responses observed in mutualistic symbiotic interactions. The intertwining of different regulatory networks revealed, shows how developmental signaling, disease symptom development, and stress signaling can be balanced

Stress knowledge map

Stress Knowledge Map (SKMhttps://skm.nib.si) is a publicly available resource containing two complementary knowledge graphs that describe the current knowledge of biochemical, signaling, and regulatory molecular interactions in plants: a highly curated model of plant stress signaling (PSS543 reactions) and a large comprehensive knowledge network (488 390 interactions). Both were constructed by domain experts through systematic curation of diverse literature and database resources. SKM provides a single entry point for investigations of plant stress response and related growth trade-offs, as well as interactive explorations of current knowledge. PSS is also formulated as a qualitative and quantitative model for systems biology and thus represents a starting point for a plant digital twin. Here, we describe the features of SKM and show, through two case studies, how it can be used for complex analyses, including systematic hypothesis generation and design of validation experiments, or to gain new insights into experimental observations in plant biology

Potato virus Y infection alters small RNA metabolism and immune response in tomato

Potato virus Y (PVY) isolate PVYC-to induces growth reduction and foliar symptoms in tomato, but new vegetation displays symptom recovery at a later stage. In order to investigate the role of micro(mi)RNA and secondary small(s)RNA-regulated mechanisms in tomato defenses against PVY, we performed sRNA sequencing from healthy and PVYC-to infected tomato plants at 21 and 30 days post-inoculation (dpi). A total of 792 miRNA sequences were obtained, among which were 123 canonical miRNA sequences, many isomiR variants, and 30 novel miRNAs. MiRNAs were mostly overexpressed in infected vs. healthy plants, whereas only a few miRNAs were underexpressed. Increased accumulation of isomiRs was correlated with viral infection. Among miRNA targets, enriched functional categories included resistance (R) gene families, transcription and hormone factors, and RNA silencing genes. Several 22-nt miRNAs were shown to target R genes and trigger the production of 21-nt phased sRNAs (phasiRNAs). Next, 500 phasiRNA-generating loci were identified, and were shown to be mostly active in PVY-infected tissues and at 21 dpi. These data demonstrate that sRNA-regulated host responses, encompassing miRNA alteration, diversification within miRNA families, and phasiRNA accumulation, regulate R and disease-responsive genes. The dynamic regulation of miRNAs and secondary sRNAs over time suggests a functional role of sRNA-mediated defenses in the recovery phenotype

Milestone M27

We present the technical report regarding the implementation of the Genialis Platform at the National Institute of Biology (NIB) within the project Hyp (J4-7636)

Transcriptional and epigenetic changes during tomato yellow leaf curl virus infection in tomato

Abstract Background Geminiviruses are DNA plant viruses that cause highly damaging diseases affecting crops worldwide. During the infection, geminiviruses hijack cellular processes, suppress plant defenses, and cause a massive reprogramming of the infected cells leading to major changes in the whole plant homeostasis. The advances in sequencing technologies allow the simultaneous analysis of multiple aspects of viral infection at a large scale, generating new insights into the molecular mechanisms underlying plant-virus interactions. However, an integrative study of the changes in the host transcriptome, small RNA profile and methylome during a geminivirus infection has not been performed yet. Using a time-scale approach, we aim to decipher the gene regulation in tomato in response to the infection with the geminivirus, tomato yellow leaf curl virus (TYLCV). Results We showed that tomato undergoes substantial transcriptional and post-transcriptional changes upon TYLCV infection and identified the main altered regulatory pathways. Interestingly, although the principal plant defense-related processes, gene silencing and the immune response were induced, this cannot prevent the establishment of the infection. Moreover, we identified extra- and intracellular immune receptors as targets for the deregulated microRNAs (miRNAs) and established a network for those that also produced phased secondary small interfering RNAs (phasiRNAs). On the other hand, there were no significant genome-wide changes in tomato methylome at 14 days post infection, the time point at which the symptoms were general, and the amount of viral DNA had reached its maximum level, but we were able to identify differentially methylated regions that could be involved in the transcriptional regulation of some of the differentially expressed genes. Conclusion We have conducted a comprehensive and reliable study on the changes at transcriptional, post-transcriptional and epigenetic levels in tomato throughout TYLCV infection. The generated genomic information is substantial for understanding the genetic, molecular and physiological changes caused by TYLCV infection in tomato

CRISPR/Cas9-mediated fine-tuning of miRNA expression in tetraploid potato

International audienceAbstract MicroRNAs (miRNAs) are small noncoding RNAs, which modulate the abundance and spatiotemporal accumulation of target mRNAs at transcriptional and post-transcriptional levels and through that play important roles in several biological processes in plants. Here we show that in polyploid species, CRISPR/Cas9 system can be used for fine-tuning of miRNA expression, which can have broader range of applications compared to knock-out mutants. We established the complete pipeline for CRISPR-Cas9-mediated modulation of miRNA expression in potato. It consists of (1) design and assembly of dual sgRNA CRISPR/Cas9 constructs, (2) transient transfection of protoplasts following fast and efficient screening by high resolution melting analysis to select functional sgRNAs, and (3) stable transformation of potato explants with functional sgRNAs and selection of regenerated transgenic lines with desired mutations and desired miRNA abundance based on sequencing and RT-qPCR. We show that miRNA-editing using dual sgRNA approach results in different types of mutations among transgenic lines but also in different alleles of the same plant, which are target site-dependent. The most frequent were short deletions, but we also detected 1-nt insertions (T or G), deletions between two sgRNAs and larger deletions. miRNA abundance correlates with the frequency and type of introduced mutations, as more extensive mutations in more alleles result in lower miRNA abundance. Interestingly, some mutated loci can generate alternative miRNAs, now novel targets were however predicted for those. In all transgenic lines with Cas9 expression, we detected mutations, suggesting high efficiency of Cas9-editing. We confirmed the miRNA-editing efficiency of our optimised approach in two different potato genotypes and three different loci