Evelyn Sánchez

Mutualistic interactions between plants and beneficial fungi rely on extensive transcriptional reprogramming in both partners, yet the underlying regulatory mechanisms coordinating these responses remain incompletely understood. Here, we combined a transcriptomics analysis with a gene regulatory network (GRN) inference to dissect the interaction between Arabidopsis thaliana and the growth-promoting fungus Trichoderma atroviride. At an early but established stage of colonization (72 h post-inoculation), we identified widespread transcriptional changes in both of the organisms, including host activation of hypoxia, stress and root development-related pathways in Arabidopsis, and fungal reprogramming of membrane transport and primary metabolism. Using DNA-binding motifs and GENIE3-based regulatory inference, we reconstructed interaction-specific GRNs for each species. The subnetworks focused on the main differentially expressed biological processes and uncovered ERF-, WRKY-, NAC- and DOF-centered modules linking hypoxia responses with developmental remodeling in the plant, whereas the putative orthologs of TFs involved in developmental and stress-related TFs in fungi, such as CrzA, RME1, NsdC, PacC and RPN4, formed a regulatory core coordinating fungal transport and metabolic adjustment. In parallel, we uncovered contrasting sRNA dynamics between the partners. While the Arabidopsis sRNA changes were limited, T. atroviride exhibited a strong induction of 20–22 nt sRNAs, including a small set of high-confidence sRNA–mRNA interactions targeting host genes involved in root function and immunity. Together, our results extend previous pathway-based descriptions of the Arabidopsis–Trichoderma mutualism and provide a systems-level, testable framework for how coordinated regulatory programs in both of the partners support the interaction.

Sulfate availability critically influences plant growth, yet the role of small RNAs, particularly microRNAs (miRNAs), in regulating responses to sulfate deficiency remains poorly understood. Here, we conducted a temporal analysis of sulfate deficiency-responsive miRNAs in the roots and leaves of Solanum lycopersicum (tomato), using an updated miRNA annotation in the SL4.0 genome. We found 40 differentially expressed miRNAs, including 2 novel, tomato-specific miRNAs. Tomato miRNAs showed an important time- and organ-specific regulation, similar to the described response of the mRNA transcriptome. Integration with transcriptomic data and Degradome-seq analysis highlighted both canonical and non-canonical targets for sulfate-responsive miRNAs. miR395, the most extensively studied miRNA, was found to control not only its conserved targets involved in sulfate transport and assimilation, but also genes involved in redox homeostasis, photosynthesis and chloride transport. Notably, most targets were repressed in leaves, suggesting miRNA-mediated downregulation of energy-intensive processes, while root targets were predominantly upregulated, including genes related to protein remodeling and antioxidant defense. Comparative analysis with Arabidopsis thaliana revealed a broader functional repertoire in tomato, suggesting species-specific adaptations to sulfate deficiency. Overall, our results underscore the critical role of miRNAs in fine-tuning organ-specific metabolic reprogramming during nutrient stress, expanding the current understanding of the regulatory landscape underlying sulfate deficiency in plants.

Sulfur is an essential element for biological molecules, with sulfate as the primary sulfur source for plants. Under sulfate-deficiency, plants undergo physiological, metabolic, transcriptional, and post-transcriptional changes. Although Solanum lycopersicum (tomato) exhibits extensive transcriptome reprogramming during sulfate deficiency, the role of microRNAs (miRNAs) in this response remains poorly explored. In this study, we analyzed miRNAs expression in tomato roots and leaves at 3- and 4-weeks post-sowing under sulfate-sufficient and sulfate-deficient conditions, leveraging a de novo annotation of miRNA genes in the SL4.0 genome. We identified several miRNAs showing tissue- and time-specific expression changes in response to sulfate deficiency, with a potential role in processes such as lignin, chlorophyll, and polysaccharides metabolism. Consistent with other plants, miR395 emerged as a key sulfur metabolism and transport regulator. Notably, we identified a novel miR395 target, SlERF2a, an Ethylene-Responsive Factor predicted to regulate numerous sulfate deficiency-responsive genes, including genes involved in plastid organization, detoxification, sulfur assimilation and starvation response. These results suggest a broader role for miR395 in sulfate-deficiency responses in tomato compared to other plants. This work provides new insights into the post-transcriptional regulation of sulfate-deficiency in tomato, providing an updated miRNA annotation for future studies on miRNA-mediated regulation in this relevant crop.

MicroRNAs (miRNAs) are small noncoding RNAs that are key players in the regulation of gene expression. In the past decade, with the increasing accessibility of high-throughput sequencing technologies, different methods have been developed to identify miRNAs, most of which rely on preexisting reference genomes. However, when a reference genome is absent or is not of high quality, such identification becomes more difficult. In this context, we developed BrumiR, an algorithm that is able to discover miRNAs directly and exclusively from small RNA (sRNA) sequencing (sRNA-seq) data. We benchmarked BrumiR with datasets encompassing animal and plant species using real and simulated sRNA-seq experiments. The results demonstrate that BrumiR reaches the highest recall for miRNA discovery, while at the same time being much faster and more efficient than the state-of-the-art tools evaluated. The latter allows BrumiR to analyze a large number of sRNA-seq experiments, from plants or animal species. Moreover, BrumiR detects additional information regarding other expressed sequences (sRNAs, isomiRs, etc.), thus maximizing the biological insight gained from sRNA-seq experiments. Additionally, when a reference genome is available, BrumiR provides a new mapping tool (BrumiR2reference) that performs an a posteriori exhaustive search to identify the precursor sequences. Finally, we also provide a machine learning classifier based on a random forest model that evaluates the sequence-derived features to further refine the prediction obtained from the BrumiR-core. The code of BrumiR and all the algorithms that compose the BrumiR toolkit are freely available at https://github.com/camoragaq/BrumiR.

In sweet cherry (Prunus avium), as in other temperate woody perennials, bud dormancy allows for survival in adverse environmental conditions during winter. During this process, environmental signals such as short days and/or low temperatures trigger internal signals that enable buds to become tolerant to the cold. The process involves tracking chilling units up to chilling the requirement fulfillment to resume growth, a transition involving transcriptional regulation, metabolic signaling, and epigenetic-related regulatory events. Massive sequencing of small RNAs was performed to identify miRNAs involved in sweet cherry dormancy by comparing their expression in field (regular seasonal) and controlled non-stop (continuous) chilling conditions. miRNAs highlighted by sequencing were validated using specific stem-loop PCR quantification, confirming expression patterns for known miRNAs such as miR156e, miR166c, miR172d, miR391, miR482c, and miR535b, as well as for newly proposed miRNAs. In silico prediction of the target genes was used to construct miRNA/target gene nodes. In particular, the involvement of the sweet cherry version for the miR156/SQUAMOSA PROMOTER-BINDING-LIKE PROTEIN genes whose expression was opposite in the two conditions suggests their involvement on dormancy regulation in sweet cherry. miRNA levels indicate that the regulation of stress-related genes and hormone synthesis modulates the expression of calcium metabolism and cell development-associated genes. Understanding the regulatory networks involved in sweet cherry dormancy, particularly in the context of miRNA involvement, represents the first step in the development of new agricultural strategies that may help overcome the increasing challenges presented by global climate change.

Botrytis cinerea and Trichoderma atroviride are two relevant fungi in agricultural systems. To gain insights into these organisms’ transcriptional gene regulatory networks (GRNs), we generated a manually curated transcription factor (TF) dataset for each of them, followed by a GRN inference utilizing available sequence motifs describing DNA-binding specificity and global gene expression data. As a proof of concept of the usefulness of this resource to pinpoint key transcriptional regulators, we employed publicly available transcriptomics data and a newly generated dual RNA-seq dataset to build context-specific Botrytis and Trichoderma GRNs under two different biological paradigms: exposure to continuous light and Botrytis-Trichoderma confrontation assays. Network analysis of fungal responses to constant light revealed striking differences in the transcriptional landscape of both fungi. On the other hand, we found that the confrontation of both microorganisms elicited a distinct set of differentially expressed genes with changes in T. atroviride exceeding those in B. cinerea. Using our regulatory network data, we were able to determine, in both fungi, central TFs involved in this interaction response, including TFs controlling a large set of extracellular peptidases in the biocontrol agent T. atroviride. In summary, our work provides a comprehensive catalog of transcription factors and regulatory interactions for both organisms. This catalog can now serve as a basis for generating novel hypotheses on transcriptional regulatory circuits in different experimental contexts.

Rapid and cost-efficient identification of Naupactus species is becoming a key process for the exportation of citrus fruit from Chile and other countries, considering the quarantine regulations for some species of the cosmopolitan genus Naupactus. This study deals with the development of a fast and sensitive detection protocol for Naupactus cervinus (Coleoptera: Curculionidae) (Boheman) and Naupactus xanthographus (Coleoptera: Curculionidae) (Germar) based on multiplex TaqMan Real-time polymerase chain reaction. Both N. cervinus and N. xanthographus primer and probe sets achieved species-specific detection in a linear range from 1 pg/μl to 1 × 10^-6 pg/μl, allowing detection of as few as 160 copies of template DNA. Non-target amplifications were not detected and a panel composed of 480 test samples had 100% coincidence with the respective morphological identification.

Background: In plants, host factors encoded by susceptibility (S) genes are indispensable for viral infection. Resistance is achieved through the impairment or the absence of those susceptibility factors. ManySgenes have been cloned from model and crop species and a majority of them are coding for members of the eukaryotic translation initiation complex, mainly eIF4E, eIF4G and their isoforms. The aim of this study was to investigate the role of those translation initiation factors in susceptibility of stone fruit species to sharka, a viral disease due toPlum pox virus(PPV).Results: For this purpose, hairpin-inducing silencing constructs based onPrunus persicaorthologs were used to generatePrunus salicina(Japanese plum) 4E and 4G silenced plants byAgrobacterium tumefaciens-mediated transformation and challenged with PPV. While down-regulatedeIFiso4Etransgenic Japanese plums were not regenerated in our conditions,eIFiso4G11-, but not theeIFiso4G10-, silenced plants displayed durable and stable resistance to PPV. We also investigated the alteration of the si- and mi-RNA profiles in transgenic and wild-type Japanese plums upon PPV infection and confirmed that the newly generated small interfering (si) RNAs, which are derived from the engineered inverted repeat construct, are the major contributor of resistance to sharka.Conclusions: Our results indicate thatSgene function of the translation initiation complex isoform is conserved inPrunusspecies. We discuss the possibilities of using RNAi silencing or loss-of-function mutations of the different isoforms of proteins involved in this complex to breed for resistance to sharka in fruit trees.

ISHS VIII International Cherry Symposium A draft genome of Prunus avium 'Karina' as a tool for genomic studies