Assessing small RNA profiles in potato diploid hybrid and its resynthesized allopolyploid reveals conserved abundance with distinct genomic distribution.

KEY MESSAGE: The shock produced by the allopolyploidization process on a potato interspecific diploid hybrid displays a non-random remobilization of the small RNAs profile on a variety of genomic features. Allopolyploidy, a complex process involving interspecific hybridization and whole genome duplication, significantly impacts plant evolution, leading to the emergence of novel phenotypes. Polyploids often present phenotypic nuances that enhance adaptability, enabling them to compete better and occasionally to colonize new habitats. Whole-genome duplication represents a genomic "shock" that can trigger genetic and epigenetic changes that yield novel expression patterns. In this work, we investigate the polyploidization effect on a diploid interspecific hybrid obtained through the cross between the cultivated potato Solanum tuberosum and the wild potato Solanum kurtzianum, by assessing the small RNAs (sRNAs) profile of the parental diploid hybrid and its derived allopolyploid. Small RNAs are key components of the epigenetic mechanisms involved in silencing by RNA-directed DNA Methylation (RdDM). A sRNA sequencing (sRNA-Seq) analysis was performed to individually profile the 21 to 22 nucleotide (21 to 22-nt) and 24-nt sRNA size classes due to their unique mechanism of biogenesis and mode of function. The composition and distribution of different genomic features and differentially accumulated (DA) sRNAs were evaluated throughout the potato genome. We selected a subset of genes associated with DA sRNAs for messenger RNA (mRNA) expression analysis to assess potential impacts on the transcriptome. Interestingly, we noted that 24-nt DA sRNAs that exclusively mapped to exons were correlated with differentially expressed mRNAs between genotypes, while this behavior was not observed when 24-nt DA sRNAs were mapped to intronic regions. These findings collectively emphasize the nonstochastic nature of sRNA remobilization in response to the genomic shock induced by allopolyploidization.

Genome-wide identification of MITE-derived microRNAs and their targets in bread wheat.

BACKGROUND: Plant miRNAs are a class of small non-coding RNAs that can repress gene expression at the post-transcriptional level by targeting RNA degradation or promoting translational repression. There is increasing evidence that some miRNAs can derive from a group of non-autonomous class II transposable elements called Miniature Inverted-repeat Transposable Elements (MITEs). RESULTS: We used public small RNA and degradome libraries from Triticum aestivum to screen for microRNAs production and predict their cleavage target sites. In parallel, we also created a comprehensive wheat MITE database by identifying novel elements and compiling known ones. When comparing both data sets, we found high homology between MITEs and 14% of all the miRNAs production sites detected. Furthermore, we show that MITE-derived miRNAs have preference for targeting degradation sites with MITE insertions in the 3' UTR regions of the transcripts. CONCLUSIONS: Our results revealed that MITE-derived miRNAs can underlay the origin of some miRNAs and potentially shape a regulatory gene network. Since MITEs are found in millions of insertions in the wheat genome and are closely linked to genic regions, this kind of regulatory network could have a significant impact on the post-transcriptional control of gene expression.

Negative modulation of SA signaling components by the capsid protein of tobacco mosaic virus is required for viral long-distance movement.

An important aspect of plant-virus interaction is the way viruses dynamically move over long distances and how plant immunity modulates viral systemic movement. Salicylic acid (SA), a well-characterized hormone responsible for immune responses against virus, is activated through different transcription factors including TGA and WRKY. In tobamoviruses, evidence suggests that capsid protein (CP) is required for long-distance movement, although its precise role has not been fully characterized yet. Previously, we showed that the CP of Tobacco Mosaic Virus (TMV)-Cg negatively modulates the SA-mediated defense. In this study, we analyzed the impact of SA-defense mechanism on the long-distance transport of a truncated version of TMV (TMV ∆CP virus) that cannot move to systemic tissues. The study showed that the negative modulation of NPR1 and TGA10 factors allows the long-distance transport of TMV ∆CP virus. Moreover, we observed that the stabilization of DELLA proteins promotes TMV ∆CP systemic movement. We also characterized a group of genes, part of a network modulated by CP, involved in TMV ∆CP long-distance transport. Altogether, our results indicate that CP-mediated downregulation of SA signaling pathway is required for the virus systemic movement, and this role of CP may be linked to its ability to stabilize DELLA proteins.

The cytosolic invertase NI6 affects vegetative growth, flowering, fruit set, and yield in tomato.

Sucrose metabolism is important for most plants, both as the main source of carbon and via signaling mechanisms that have been proposed for this molecule. A cleaving enzyme, invertase (INV) channels sucrose into sink metabolism. Although acid soluble and insoluble invertases have been largely investigated, studies on the role of neutral invertases (A/N-INV) have lagged behind. Here, we identified a tomato A/N-INV encoding gene (NI6) co-localizing with a previously reported quantitative trait locus (QTL) largely affecting primary carbon metabolism in tomato. Of the eight A/N-INV genes identified in the tomato genome, NI6 mRNA is present in all organs, but its expression was higher in sink tissues (mainly roots and fruits). A NI6-GFP fusion protein localized to the cytosol of mesophyll cells. Tomato NI6-silenced plants showed impaired growth phenotype, delayed flowering and a dramatic reduction in fruit set. Global gene expression and metabolite profile analyses of these plants revealed that NI6 is not only essential for sugar metabolism, but also plays a signaling role in stress adaptation. We also identified major hubs, whose expression patterns were greatly affected by NI6 silencing; these hubs were within the signaling cascade that coordinates carbohydrate metabolism with growth and development in tomato.

Vineyard environments influence Malbec grapevine phenotypic traits and DNA methylation patterns in a clone-dependent way.

KEY MESSAGE: By studying three cv. Malbec clones cultivated in two vineyards with contrasting environmental conditions, we demonstrated that DNA methylation has an important role in the phenotypic plasticity and that epigenetic modulation is clone-dependent. Clonal selection and vegetative propagation determine low genetic variability in grapevine cultivars, although it is common to observe diverse phenotypes. Environmental signals may induce epigenetic changes altering gene expression and phenotype. The range of phenotypes that a genotype expresses in different environments is known as phenotypic plasticity. DNA methylation is the most studied epigenetic mechanism, but only few works evaluated this novel source of variability in grapevines. In the present study, we analyzed the effects on phenotypic traits and epigenome of three Vitis vinifera cv. Malbec clones cultivated in two contrasting vineyards of Mendoza, Argentina. Anonymous genome regions were analyzed using methylation-sensitive amplified polymorphism (MSAP) markers. Clone-dependent phenotypic and epigenetic variability between vineyards were found. The clone that presented the clearer MSAP differentiation between vineyards was selected and analyzed through reduced representation bisulfite sequencing. Twenty-nine differentially methylated regions between vineyards were identified and associated to genes and/or promoters. We discuss about a group of genes related to hormones homeostasis and sensing that could provide a hint of the epigenetic role in the determination of the different phenotypes observed between vineyards and conclude that DNA methylation has an important role in the phenotypic plasticity and that epigenetic modulation is clone-dependent.

RdDM pathway components differentially modulate Tobamovirus symptom development.

KEY MESSAGE: The crop yield losses induced by phytoviruses are mainly associated with the symptoms of the disease. DNA modifications as methylation can modulate the information coded by the sequence, process named epigenetics. Viral infection can change the expression patterns of different genes linked to defenses and symptoms. This work represents the initial step to expose the role of epigenetic process, in the production of symptoms associated with plants-virus interactions. Small RNAs (sRNAs) are important molecules for gene regulation in plants and play an essential role in plant-pathogen interactions. Researchers have evaluated the relationship between viral infections as well as the endogenous accumulation of sRNAs and the transcriptional changes associated with the production of symptoms, but little is known about a possible direct role of epigenetics, mediated by 24-nt sRNAs, in the induction of these symptoms. Using different RNA directed DNA methylation (RdDM) pathway mutants and a triple demethylase mutant; here we demonstrate that the disruption of RdDM pathway during viral infection produce alterations in the plant transcriptome and in consequence changes in plant symptoms. This study represents the initial step in exposing that DNA methylation directed by endogenous sRNAs has an important role, uncoupled to defense, in the production of symptoms associated with plant-virus interactions.

Genomic re-assessment of the transposable element landscape of the potato genome.

KEY MESSAGE: We provide a comprehensive and reliable potato TE landscape, based on a wide variety of identification tools and integrative approaches, producing clear and ready-to-use outputs for the scientific community. Transposable elements (TEs) are DNA sequences with the ability to autoreplicate and move throughout the host genome. TEs are major drivers in stress response and genome evolution. Given their significance, the development of clear and efficient TE annotation pipelines has become essential for many species. The latest de novo TE discovery tools, along with available TEs from Repbase and sRNA-seq data, allowed us to perform a reliable potato TEs detection, classification and annotation through an open-source and freely available pipeline ( https://github.com/DiegoZavallo/TE_Discovery ). Using a variety of tools, approaches and rules, we were able to provide a clearly annotated of characterized TEs landscape. Additionally, we described the distribution of the different types of TEs across the genome, where LTRs and MITEs present a clear clustering pattern in pericentromeric and subtelomeric/telomeric regions respectively. Finally, we analyzed the insertion age and distribution of LTR retrotransposon families which display a distinct pattern between the two major superfamilies. While older Gypsy elements concentrated around heterochromatic regions, younger Copia elements located predominantly on euchromatic regions. Overall, we delivered not only a reliable, ready-to-use potato TE annotation files, but also all the necessary steps to perform de novo detection for other species.

MITE Tracker: an accurate approach to identify miniature inverted-repeat transposable elements in large genomes.

BACKGROUND: Miniature inverted-repeat transposable elements (MITEs) are short, non-autonomous class II transposable elements present in a high number of conserved copies in eukaryote genomes. An accurate identification of these elements can help to shed light on the mechanisms controlling genome evolution and gene regulation. The structure and distribution of these elements are well-defined and therefore computational approaches can be used to identify MITEs sequences. RESULTS: Here we describe MITE Tracker, a novel, open source software program that finds and classifies MITEs using an efficient alignment strategy to retrieve nearby inverted-repeat sequences from large genomes. This program groups them into high sequence homology families using a fast clustering algorithm and finally filters only those elements that were likely transposed from different genomic locations because of their low scoring flanking sequence alignment. CONCLUSIONS: Many programs have been proposed to find MITEs hidden in genomes. However, none of them are able to process large-scale genomes such as that of bread wheat. Furthermore, in many cases the existing methods perform high false-positive rates (or miss rates). The rice genome was used as reference to compare MITE Tracker against known tools. Our method turned out to be the most reliable in our tests. Indeed, it revealed more known elements, presented the lowest false-positive number and was the only program able to run with the bread wheat genome as input. In wheat, MITE Tracker discovered 6013 MITE families and allowed the first structural exploration of MITEs in the complete bread wheat genome.

Immune receptor genes and pericentromeric transposons as targets of common epigenetic regulatory elements.

Pattern recognition receptors (PRR) and nucleotide-binding leucine-rich repeat proteins (NLR) are major components of the plant immune system responsible for pathogen detection. To date, the transcriptional regulation of PRR/NLR genes is poorly understood. Some PRR/NLR genes are affected by epigenetic changes of neighboring transposable elements (TEs) (cis regulation). We analyzed whether these genes can also respond to changes in the epigenetic marks of distal pericentromeric TEs (trans regulation). We found that Arabidopsis tissues infected with Pseudomonas syringae pv. tomato (Pst) initially induced the expression of pericentromeric TEs, and then repressed it by RNA-directed DNA methylation (RdDM). The latter response was accompanied by the accumulation of small RNAs (sRNAs) mapping to the TEs. Curiously these sRNAs also mapped to distal PRR/NLR genes, which were controlled by RdDM but remained induced in the infected tissues. Then, we used non-infected mom1 (Morpheus' molecule 1) mutants that expressed pericentromeric TEs to test if they lose repression of PRR/NLR genes. mom1 plants activated several PRR/NLR genes that were unlinked to MOM1-targeted TEs, and showed enhanced resistance to Pst. Remarkably, the increased defenses of mom1 were abolished when MOM1/RdDM-mediated pericentromeric TEs silencing was re-established. Therefore, common sRNAs could control PRR/NLR genes and distal pericentromeric TEs and preferentially silence TEs when they are activated.

Mal de Río Cuarto Virus Infection Triggers the Production of Distinctive Viral-Derived siRNA Profiles in Wheat and Its Planthopper Vector.

Plant reoviruses are able to multiply in gramineae plants and delphacid vectors encountering different defense strategies with unique features. This study aims to comparatively assess alterations of small RNA (sRNA) populations in both hosts upon virus infection. For this purpose, we characterized the sRNA profiles of wheat and planthopper vectors infected by Mal de Río Cuarto virus (MRCV, Fijivirus, Reoviridae) and quantified virus genome segments by quantitative reverse transcription PCR We provide evidence that plant and insect silencing machineries differentially recognize the viral genome, thus giving rise to distinct profiles of virus-derived small interfering RNAs (vsiRNAs). In plants, most of the virus genome segments were targeted preferentially within their upstream sequences and vsiRNAs mapped with higher density to the smaller genome segments than to the medium or larger ones. This tendency, however, was not observed in insects. In both hosts, vsiRNAs were equally derived from sense and antisense RNA strands and the differences in vsiRNAs accumulation did not correlate with mRNAs accumulation. We also established that the piwi-interacting RNA (piRNA) pathway was active in the delphacid vector but, contrary to what is observed in virus-infected mosquitoes, virus-specific piRNAs were not detected. This work contributes to the understanding of the silencing response in insect and plant hosts.

TMV induces RNA decay pathways to modulate gene silencing and disease symptoms.

RNA decay pathways comprise a combination of RNA degradation mechanisms that are implicated in gene expression, development and defense responses in eukaryotes. These mechanisms are known as the RNA Quality Control or RQC pathways. In plants, another important RNA degradation mechanism is the post-transcriptional gene silencing (PTGS) mediated by small RNAs (siRNAs). Notably, the RQC pathway antagonizes PTGS by preventing the entry of dysfunctional mRNAs into the silencing pathway to avoid global degradation of mRNA by siRNAs. Viral transcripts must evade RNA degrading mechanisms, thus viruses encode PTGS suppressor proteins to counteract viral RNA silencing. Here, we demonstrate that tobacco plants infected with TMV and transgenic lines expressing TMV MP and CP (coat protein) proteins (which are not linked to the suppression of silencing) display increased transcriptional levels of RNA decay genes. These plants also showed accumulation of cytoplasmic RNA granules with altered structure, increased rates of RNA decay for transgenes and defective transgene PTGS amplification. Furthermore, knockdown of RRP41 or RRP43 RNA exosome components led to lower levels of TMV accumulation with milder symptoms after infection, several developmental defects and miRNA deregulation. Thus, we propose that TMV proteins induce RNA decay pathways (in particular exosome components) to impair antiviral PTGS and this defensive mechanism would constitute an additional counter-defense strategy that lead to disease symptoms.

Differential mRNA Accumulation upon Early Arabidopsis thaliana Infection with ORMV and TMV-Cg Is Associated with Distinct Endogenous Small RNAs Level.

Small RNAs (sRNAs) play important roles in plant development and host-pathogen interactions. Several studies have highlighted the relationship between viral infections, endogenous sRNA accumulation and transcriptional changes associated with symptoms. However, few studies have described a global analysis of endogenous sRNAs by comparing related viruses at early stages of infection, especially before viral accumulation reaches systemic tissues. An sRNA high-throughput sequencing of Arabidopsis thaliana leaf samples infected either with Oilseed rape mosaic virus (ORMV) or crucifer-infecting Tobacco mosaic virus (TMV-Cg) with slightly different symptomatology at two early stages of infection (2 and 4 dpi) was performed. At early stages, both viral infections strongly alter the patterns of several types of endogenous sRNA species in distal tissues with no virus accumulation suggesting a systemic signaling process foregoing to virus spread. A correlation between sRNAs derived from protein coding genes and the associated mRNA transcripts was also detected, indicating that an unknown recursive mechanism is involved in a regulatory circuit encompassing this sRNA/mRNA equilibrium. This work represents the initial step in uncovering how differential accumulation of endogenous sRNAs contributes to explain the massive alteration of the transcriptome associated with plant-virus interactions.

TMV-Cg Coat Protein stabilizes DELLA proteins and in turn negatively modulates salicylic acid-mediated defense pathway during Arabidopsis thaliana viral infection.

BACKGROUND: Plant viral infections disturb defense regulatory networks during tissue invasion. Emerging evidence demonstrates that a significant proportion of these alterations are mediated by hormone imbalances. Although the DELLA proteins have been reported to be central players in hormone cross-talk, their role in the modulation of hormone signaling during virus infections remains unknown. RESULTS: This work revealed that TMV-Cg coat protein (CgCP) suppresses the salicylic acid (SA) signaling pathway without altering defense hormone SA or jasmonic acid (JA) levels in Arabidopsis thaliana. Furthermore, it was observed that the expression of CgCP reduces plant growth and delays the timing of floral transition. Quantitative RT-qPCR analysis of DELLA target genes showed that CgCP alters relative expression of several target genes, indicating that the DELLA proteins mediate transcriptional changes produced by CgCP expression. Analyses by fluorescence confocal microscopy showed that CgCP stabilizes DELLA proteins accumulation in the presence of gibberellic acid (GA) and that the DELLA proteins are also stabilized during TMV-Cg virus infections. Moreover, DELLA proteins negatively modulated defense transcript profiles during TMV-Cg infection. As a result, TMV-Cg accumulation was significantly reduced in the quadruple-DELLA mutant Arabidopsis plants compared to wild type plants. CONCLUSIONS: Taken together, these results demonstrate that CgCP negatively regulates the salicylic acid-mediated defense pathway by stabilizing the DELLA proteins during Arabidopsis thaliana viral infection, suggesting that CgCP alters the stability of DELLAs as a mechanism of negative modulation of antiviral defense responses.

Salicylic acid determines differential senescence produced by two Turnip mosaic virus strains involving reactive oxygen species and early transcriptomic changes.

Losses produced by virus diseases depend mostly on symptom severity. Turnip mosaic virus (TuMV) is one of the most damaging and widespread potyvirus infecting members of the family Brassicaceae, including Arabidopsis thaliana. We used JPN1 and UK1 TuMV strains to characterize viral infections regarding symptom development, senescence progression, antioxidant response, reactive oxygen species (ROS) accumulation, and transcriptional profiling. Both isolates, despite accumulating similar viral titers, induced different symptomatology and strong differences in oxidative status. Early differences in several senescence-associated genes linked to the ORE1 and ORS1 regulatory networks as well as persistent divergence in key ROS production and scavenging systems of the plant were detected. However, at a later stage, both strains induced nutrient competition, indicating that senescence rates are influenced by different mechanisms upon viral infections. Analyses of ORE1 and ORS1 levels in infected Brassica juncea plants showed a similar pattern, suggesting a conserved differential response to both strains in Brassicaceae spp. Transcriptional analysis of the ORE1 and ORS1 regulons showed similarities between salicylic acid (SA) response and the early induction triggered by UK1, the most severe strain. By means of SA-defective NahG transgenic plants, we found that differential senescence progression and ROS accumulation between strains rely on an intact SA pathway.

Isolation and functional characterization of two novel seed-specific promoters from sunflower (Helianthus annuus L.).

The promoter region of two sunflower (Helianthus annuus L. HA89 genotype) seed specifically expressed genes, coding for an oleate desaturase (HaFAD2-1) and a lipid transfer protein (HaAP10), were cloned and in silico characterized. The isolated fragments are 867 and 964 bp long, respectively, and contain several seed-specific motifs, such as AACA motif, ACGT element, E-Boxes, SEF binding sites and GCN4 motif. Functional analysis of these promoters in transgenic Arabidopsis plants was investigated after fusing them with the beta-glucuronidase (GUS) reporter gene. None of the promoters triggered GUS activity in any vegetative tissue, with the exception of early seedling cotyledons. HaFAD2-1 and HaAP10 promoters were tested along seed development from globular stage to mature seeds. GUS staining was restricted to embryonic tissue and quantitative fluorometric assays showed high activity values at the later stages of development. In this work we demonstrate that HaFAD2-1 promoter is as strong as 35S promoter even though it is a tissue-specific promoter and its activity derived just from the embryo, thus confirming that it can be considered a strong highly specific seed promoter useful for biotechnology applications.

Rooting in Km selective media as efficient in vitro selection method for sunflower genetic transformation

Despite of numerous publications in sunflower genetic transformation, there is no efficient or reproducible protocol with low number of escapes. The latter would indicate that the selection method is not effective. In this work we used Km as selective agent, Agrobacterium tumefaciens EHA105 strain and a vector with the nptII gene under the nos promoter and uidA gene under 35S promoter. The response of agroinfected (A) and control (C) explants during the in vitro culture was studied and in both cases in presence or absence of Km in order to assign a differential morphologic response between transformed and non-transformed plants. The characteristics analyzed were: height, colour/aspect of the plantlets, in vitro rooting and in vitro bud-flower development. Selection was applied from the third regeneration media. Among the A plantlets two were capable of rooting, being positive by PCR, whereas the C were unable to root in presence of Km. One of them gave 6 seeds and in these plants, it was determined the presence of the transgene by PCR and GUS staining. This work shows that in Km selection, colour/aspect of shoots is not useful as selection criteria whereas rooting is an effective selection method in which no escapes were obtained.