Edoardo Bertolini

Molecular and Physiological Analysis of Growth-Limiting Drought Stress in Brachypodium distachyon Leaves

The drought-tolerant grass Brachypodium distachyon is an emerging model species for temperate grasses and cereal crops. To explore the usefulness of this species for drought studies, a reproducible in vivo drought assay was developed. Spontaneous soil drying led to a 45% reduction in leaf size, and this was mostly due to a decrease in cell expansion, whereas cell division remained largely unaffected by drought. To investigate the molecular basis of the observed leaf growth reduction, the third Brachypodium leaf was dissected in three zones, namely proliferation, expansion, and mature zones, and subjected to transcriptome analysis, based on a whole-genome tiling array. This approach allowed us to highlight that transcriptome profiles of different developmental leaf zones respond differently to drought. Several genes and functional processes involved in drought tolerance were identified. The transcriptome data suggest an increased energy availability in the proliferation zones, along with an up-regulation of sterol synthesis that may influence membrane fluidity. This information may be used to improve the tolerance of temperate cereals to drought, which is undoubtedly one of the major environmental challenges faced by agriculture today and in the near future.

Addressing the role of microRNAs in reprogramming leaf growth during drought stress in Brachypodium distachyon

SUMMARY We investigated the role of known and newly discovered miRNAs in drought response and leaf development in Brachypodium distachyon. Differential expression analyses and miRNA-target predictions suggest evidence for regulatory networks controlling cell division and expansion in normal and stressed conditions.

miRVine: a microRNA expression atlas of grapevine based on small RNA sequencing

BackgroundmiRNAs are the most abundant class of small non-coding RNAs, and they are involved in post-transcriptional regulations, playing a crucial role in the refinement of genetic programming during plant development. Here we present a comprehensive picture of miRNA regulation in Vitis vinifera L. plant during its complete life cycle. Furthering our knowledge about the post-transcriptional regulation of plant development is fundamental to understand the biology of such an important crop.ResultsWe analyzed 70 small RNA libraries, prepared from berries, inflorescences, tendrils, buds, carpels, stamens and other samples at different developmental stages. One-hundred and ten known and 175 novel miRNAs have been identified and a wide grapevine expression atlas has been described. The distribution of miRNA abundance reveals that 22 novel miRNAs are specific to stamen, and two of them are, interestingly, involved in ethylene biosynthesis, while only few miRNAs are highly specific to other organs. Thirty-eight miRNAs are present in all our samples, suggesting a role in key regulatory circuit. On the basis of miRNAs abundance and distribution across samples and on the estimated correlation, we suggest that miRNA expression define organ identity. We performed target prediction analysis and focused on miRNA expression analysis in berries and inflorescence during their development, providing an initial functional description of the identified miRNAs.ConclusionsOur findings represent a very extensive miRNA expression atlas in grapevine, allowing the definition of how the spatio-temporal distribution of miRNAs defines organ identity. We describe miRNAs abundance in specific tissues not previously described in grapevine and contribute to future targeted functional analyses. Finally, we present a deep characterization of miRNA involvement in berry and inflorescence development, suggesting a role for miRNA-driven hormonal regulation.

Draft Whole-Genome Sequence of the Biocontrol Agent Trichoderma harzianum T6776

ABSTRACT Trichoderma harzianum T6776 is a promising beneficial isolate whose effects consist of growth promotion, positive response of photosynthetic activity, hormonal signaling, and carbon partitioning in tomato, coupled with biocontrol of plant pathogens. Here, we present the first genome assembly of T6776, providing a useful platform for the scientific community.

microRNAs differentially modulated in response to heat and drought stress in durum wheat cultivars with contrasting water use efficiency

Plant stress response is a complex molecular process based on transcriptional and posttranscriptional regulation of many stress-related genes. microRNAs are the best-studied class of small RNAs known to play key regulatory roles in plant response to stress, besides being involved in plant development and organogenesis. We analyzed the leaf miRNAome of two durum wheat cultivars (Cappelli and Ofanto) characterized by a contrasting water use efficiency, exposed to heat stress, and mild and severe drought stress. On the whole, we identified 98 miRNA highly similar to previously known miRNAs and grouped in 47 MIR families, as well as 85 novel candidate miRNA, putatively wheat specific. A total of 80 known and novel miRNA precursors were found differentially expressed between the two cultivars or modulated by stress and many of them showed a cultivar-specific expression profile. Interestingly, most in silico predicted targets of the miRNAs coming from the differentially expressed precursors have been experimentally linked in other species to mechanisms controlling stomatal movement, a finding in agreement with previous results showing that Cappelli has a lower stomatal conductance than Ofanto. Selected miRNAs were validated through a standardized and reliable stem-loop qRT-PCR procedure.

Identification and characterization of durum wheat microRNAs in leaf and root tissues

MicroRNAs are a class of post-transcriptional regulators of plant developmental and physiological processes and responses to environmental stresses. Here, we present the study regarding the annotation and characterization of MIR genes conducted in durum wheat. We characterized the miRNAome of leaf and root tissues at tillering stage under two environmental conditions: irrigated with 100% (control) and 55% of evapotranspiration (early water stress). In total, 90 microRNAs were identified, of which 32 were classified as putative novel and species-specific miRNAs. In addition, seven microRNA homeologous groups were identified in each of the two genomes of the tetraploid durum wheat. Differential expression analysis highlighted a total of 45 microRNAs significantly differentially regulated in the pairwise comparisons leaf versus root. The miRNA families, miR530, miR395, miR393, miR5168, miR396 and miR166, miR171, miR319, and miR167, were the most expressed in leaves in comparison to roots. Putative microRNA targets were predicted for both five and three prime sequences derived from the stem-loop of the MIR gene. Gene ontology analysis showed significant overrepresented gene categories in microRNA targets belonging to transcription factors, phenylpropanoids, oxydases, and lipid binding-protein. This work represents one of the first genome wide characterization of MIR genes in durum wheat, identifying leaf and root tissue-specific microRNAs. This genomic identification of microRNAs together with the analysis of their expression profiles is a well-accepted starting point leading to a better comprehension of the role of MIR genes in the genus Triticum.

Long noncoding RNAs in the model species Brachypodium distachyon

Eukaryotic genomes are pervasively transcribed and only a small portion of the transcribed sequences belongs to protein coding genes. High-throughput sequencing technology contributed to consolidate this perspective, allowing the identification of numerous noncoding RNAs with key roles in biological processes. Long noncoding RNAs (lncRNAs) are transcripts longer than 200 nt with limited phylogenetic conservation, expressed at low levels and characterized by tissue/organ specific expression profiles. Although a large set of lncRNAs has been identified, the functional roles of lncRNAs are only beginning to be recognized and the molecular mechanism of lncRNA-mediated gene regulation remains largely unexplored, particularly in plants where their annotation and characterization are still incomplete. Using public and proprietary poly-(A)+ RNA-seq data as well as a collection of full length ESTs from several organs, developmental stages and stress conditions in three Brachypodium distachyon inbred lines, we describe the identification and the main features of thousands lncRNAs. Here we provide a genome-wide characterization of lncRNAs, highlighting their intraspecies conservation and describing their expression patterns among several organs/tissues and stress conditions. This work represents a fundamental resource to deepen our knowledge on long noncoding RNAs in C3 cereals, allowing the Brachypodium community to exploit these results in future research programs.

Long non-coding RNAs in wild wheat progenitors

Triticum urartu and Aegilops tauschii are the diploid progenitors of the hexaploid Triticum aestivum (AuAuBBDD), donors of the Au and D genome respectively. In this work we investigate the long noncoding RNAs (lncRNAs) component of the genomes of these two wild wheat relatives. Sixty-eight RNA-seq libraries generated from several organs and conditions were retrieved from public databases. We annotated and characterized 14,515 T. urartu and 20,908 Ae. tauschii bona-fide lncRNA transcripts that show features similar to those of other plant and animal counterparts. Thousands of lncRNAs were found significantly modulated in different organs and exhibited organ specific expression, with a predominant accumulation in the spike, fostering the hypothesis of their crucial role in reproductive organs. Most of the organ-specific lncRNAs were found associated with transposable elements (TEs), indicating the possible role of TEs in lncRNA origin, differentiation and function. The majority of T. urartu and Ae. tauschii lncRNAs appear to be species-specific; nevertheless, we found some lncRNAs conserved between the two wheat progenitors, highlighting the presence and conservation of exonic splicing enhancers sites in multi-exon conserved lncRNAs. In addition, we found cases of lncRNA conservation and their cis regulatory regions spanning the wheat pre-domestication and post-domestication period. Altogether, these results represent the first comprehensive genome-wide encyclopedia of lncRNAs in wild wheat relatives, and they provide clues as to the hidden regulatory pathway mediated by long noncoding RNAs in these largely unexplored wheat progenitors.

Comparative transcriptome analysis of two citrus germplasms with contrasting susceptibility to Phytophthora nicotianae provides new insights into tolerance mechanisms

Key messageHost perception of Phytophthora nicotianae switching to necrotrophy is fundamental for disease tolerance of citrus. It involves an HR-like response, strengthening of the cell wall structure and hormonal signaling.AbstractStem rot caused by P. nicotianae is a worldwide disease of several important crops, including citrus. Given the growing awareness of chemical fungicides drawbacks, genetic improvement of citrus rootstocks remains the best alternative. However, the molecular basis underlying the successful response of resistant and/or tolerant genotypes remains poorly understood. Therefore, we performed a transcriptomic analysis to examine the differential defense response to P. nicotianae of two germplasms—tolerant sour orange (SO, Citrus aurantium) and susceptible Madam Vinous (MV, C. sinensis)—in both the biotrophic and necrotrophic phases of host–pathogen interaction. Our results revealed the necrotrophic phase as a decisive turning point, since it included stronger modulation of a number of genes implicated in pathogen perception, signal transduction, HR-like response, transcriptional reprogramming, hormone signaling, and cell wall modifications. In particular, the pathogen perception category reflected the ability of SO to perceive the pathogen even after its switch to necrotrophy, and thus to cope successfully with the infection, while MV failed. The concomitant changes in genes involved in the remaining functional categories seemed to prevent pathogen spread. This investigation provided further understanding of the successful defense mechanisms of C. aurantium against P. nicotianae, which might be exploited in post-genomic strategies to develop resistant Citrus genotypes.

Transcriptome analysis of Phoenix canariensis Chabaud in response to Rhynchophorus ferrugineus Olivier attacks.

Red Palm Weevil (RPW, Rhynchophorus ferrugineus Olivier) threatens most palm species worldwide. Until now, no studies have analyzed the gene regulatory networks of Phoenix canariensis (Chabaud) in response to RPW attacks. The aim of this study was to fill this knowledge gap. Providing this basic knowledge is very important to improve its management. Results: A deep transcriptome analysis was performed on fully expanded leaves of healthy non-infested trees and attacked trees at two symptom stages (middle and late infestation). A total of 54 genes were significantly regulated during middle stage. Pathway enrichment analysis showed that phenylpropanoid-related pathways were induced at this stage. More than 3300 genes were affected during late stage of attacks. Higher transcript abundances were observed for lipid fatty acid metabolism (fatty acid and glycerolipids), tryptophan metabolism, phenylpropanoid metabolism. Key RPW-modulated genes involved in innate response mediated by hormone crosstalk were observed belonging to auxin, jasmonate and salicylic acid (SA) pathways. Among transcription factors, some WRKYs were clearly induced. qRT-PCR validation confirmed the upregulation of key genes chosen as validation of transcriptomic analysis. Conclusion: A subset of these genes may be further analyzed in future studies to confirm their specificity to be induced by RPW infestations.