miRNA Profiling and Its Role in Multi-Omics Regulatory Networks Connected with Somaclonal Variation in Cucumber (Cucumis sativus L.).

The role of miRNAs in connection with the phenomenon of somaclonal variation, which occurs during plant in vitro culture, remains uncertain. This study aims to investigate the possible role of miRNAs in multi-omics regulatory pathways in cucumber somaclonal lines. For this purpose, we performed sRNA sequencing (sRNA-seq) from cucumber fruit samples identified 8, 10 and 44 miRNAs that are differentially expressed between somaclones (S1, S2, S3 lines) and the reference B10 line of Cucumis sativus. For miRNA identification, we use ShortStack software designed to filter miRNAs from sRNAs according to specific program criteria. The identification of predicted in-silico targets revealed 2,886 mRNAs encoded by 644 genes. The functional annotation of miRNA's target genes and gene ontology classification revealed their association with metabolic processes, response to stress, multicellular organism development, biosynthetic process and catalytic activity. We checked with bioinformatic analyses for possible interactions at the level of target proteins, differentially expressed genes (DEGs) and genes affected by genomic polymorphisms. We assume that miRNAs can indirectly influence molecular networks and play a role in many different regulatory pathways, leading to somaclonal variation. This regulation is supposed to occur through the process of the target gene cleavage or translation inhibition, which in turn affects the proteome, as we have shown in the example of molecular networks. This is a new approach combining levels from DNA-seq through mRNA-seq, sRNA-seq and in silico PPI in the area of plants' somaclonal variation.

Profiling the Proteome of Cyst Nematode-Induced Syncytia on Tomato Roots.

Cyst nematodes are important herbivorous pests in agriculture that obtain nutrients through specialized root structures termed syncytia. Syncytium initiation, development, and functioning are a research focus because syncytia are the primary interface for molecular interactions between the host plant and parasite. The small size and complex development (over approximately two weeks) of syncytia hinder precise analyses, therefore most studies have analyzed the transcriptome of infested whole-root systems or syncytia-containing root segments. Here, we describe an effective procedure to microdissect syncytia induced by Globodera rostochiensis from tomato roots and to analyze the syncytial proteome using mass spectrometry. As little as 15 mm2 of 10-µm-thick sections dissected from 30 syncytia enabled the identification of 100-200 proteins in each sample, indicating that mass-spectrometric methods currently in use achieved acceptable sensitivity for proteome profiling of microscopic samples of plant tissues (approximately 100 µg). Among the identified proteins, 48 were specifically detected in syncytia and 7 in uninfected roots. The occurrence of approximately 50% of these proteins in syncytia was not correlated with transcript abundance estimated by quantitative reverse-transcription PCR analysis. The functional categories of these proteins confirmed that protein turnover, stress responses, and intracellular trafficking are important components of the proteome dynamics of developing syncytia.

The suppression of tomato defence response genes upon potato cyst nematode infection indicates a key regulatory role of miRNAs.

Potato cyst nematode Globodera rostochiensis is an obligate parasite of solanaceous plants, triggering metabolic and morphological changes in roots which may result in substantial crop yield losses. Previously, we used the cDNA-AFLP to study the transcriptional dynamics in nematode infected tomato roots. Now, we present the rescreening of already published, upregulated transcript-derived fragment dataset using the most current tomato transcriptome sequences. Our reanalysis allowed to add 54 novel genes to 135, already found as upregulated in tomato roots upon G. rostochiensis infection (in total - 189). We also created completely new catalogue of downregulated sequences leading to the discovery of 76 novel genes. Functional classification of candidates showed that the 'wound, stress and defence response' category was enriched in the downregulated genes. We confirmed the transcriptional dynamics of six genes by qRT-PCR. To place our results in a broader context, we compared the tomato data with Arabidopsis thaliana, revealing similar proportions of upregulated and downregulated genes as well as similar enrichment of defence related transcripts in the downregulated group. Since transcript suppression is quite common in plant-nematode interactions, we assessed the possibility of miRNA-mediated inverse correlation on several tomato sequences belonging to NB-LRR and receptor-like kinase families. The qRT-PCR of miRNAs and putative target transcripts showed an opposite expression pattern in 9 cases. These results together with in silico analyses of potential miRNA targeting to the full repertoire of tomato R-genes show that miRNA mediated gene suppression may be a key regulatory mechanism during nematode parasitism.