Livia Donaire

Tomato brown rugose fruit virus (Tobamovirus fructirugosum, ToBRFV) is an emerging virus that affects tomatoes, capsicum, and chili. Since its first detection in Jordan in 2015, the virus has been reported in more than 40 countries across all the continents. In Morocco, the virus was reported for the first time in October 2021. However, its genetic diversity remains unexplored. In this work, we used a collection of 100 tomato fruits from local markets to investigate the virus’s variability. Thirty-eight sequences were recovered and used to study evolutionary pressures acting on the N-terminus of the RNA-dependent RNA polymerase, the movement protein (MP), and the coat protein (CP) genes. The genetic diversity among Moroccan sequences was low, with over 99% nucleotide identity, which is consistent with the global situation, with the CP exhibiting higher diversity followed by the MP. We identified two sites with non-synonymous substitutions in the CP, and one in the MP. We used haplotype network analyses to reveal the population structure within the Moroccan isolates and studied their relationships with sequences from the rest of the world. Sequences from Morocco showed a clear geographic structure, suggesting that geographic factors, potentially combined with agricultural practices, may contribute to shaping the population structure of ToBRFV in Morocco. Our analyses suggest few introduction events, probably from Israel, Jordan, and Italy.

Tomato brown rugose fruit virus ( Tobamovirus fructirugosum ) is an emerging virus that affects tomatoes, capsicum, and chili. Since its first detection in Jordan in 2015, the virus was reported in more than 40 countries across all the continents. In Morocco, the virus was reported for the first time in October 2021. However, its genetic diversity remains unexplored. In this work, we used a collection of tomato fruits from local markets to investigate the variability of the virus in the country. We explored the different pressures acting on the N-terminus of the RNA-dependent RNA polymerase, the movement protein, and the coat protein genes. Then, we used haplotype network analyses to reveal the population structure within the Moroccan isolates and studied their relationships with the ones from the world. We found that genetic diversity is low, which is consistent with the global situation. No signatures of diversifying selection were detected across the analyzed genes. However, the virus sequences from Morocco showed a clear geographic structure, suggesting that geographic factors probably combined with agricultural practices may contribute to shaping the population structure of ToBRFV in Morocco.

Cucumber green mottle mosaic virus (CGMMV) is a tobamovirus that causes a disease affecting cucumber crops worldwide, leading to significant economic losses. To study the variability of CGMMV in southeastern Spain, partial genome sequences were obtained from isolates collected in 2017 and 2020 from cucumber crops. Phylogenetic analyses revealed that isolates clustered into two major groups, Asian (AS)-like and European (EU)-like CGMMV isolates. These two groups coexisted in the same area, crops, and even individual plants, although the AS type predominated. The accumulation and symptom expression of molecularly cloned isolates from these two groups were assessed in two cucumber cultivars (resistant and susceptible) under both summer and winter conditions. An in planta antagonistic interaction was detected between the AS and EU isolates, in which the accumulation of CGMMV-EU was suppressed during mixed infections. A multivariate analysis did not identify statistically significant differences due to variations in environmental conditions. Unlike CGMMV-EU, CGMMV-AS did not show significant differences in accumulation based on the plant genotype. To further investigate this, the response to CGMMV-AS infection was analyzed in additional susceptible and resistant cultivars. All cultivars appeared to be similarly susceptible to CGMMV-AS, in contrast to CGMMV-EU, which accumulated to a much lower extent in resistant compared to susceptible plants. These results reinforce the need to continue epidemiological surveillance, identify new sources of resistance, and implement strict control of infected seed trade—given the growing threat that CGMMV-AS isolates pose to cucumber cultivation.

Taxonomy: Pepino mosaic virus (PepMV); species: Potexvirus pepini; family: Alphaflexiviridae ; order: Tymovirales . Virion and Genome Properties: PepMV virions are rod‐shaped, non‐enveloped, and flexuous particles. They encapsidate a monopartite, single‐stranded, positive‐sense RNA genome of approximately 6.4 kb, that encodes, in the sense orientation, a replicase, three triple gene block proteins, and the coat protein. Host Range: PepMV is widespread and causes a major disease in intensive tomato crops. Natural infections have also been reported in kachuma (also known as cachum or pepino dulce ; Solanum muricatum ), basil, wild tomato species, and weeds from various families growing near affected tomato crops. Experimentally, PepMV can infect plants of virtually all Solanaceae species. Diversity: Seven genetic types of PepMV are currently recognised: the original Peruvian (LP), European (EU), American US1, American US2, Chilean (CH2), the new Peruvian (PES), and Europe and the Asia‐Pacific region (EAP). Transmission: PepMV is efficiently transmitted through mechanical means and shows a low rate of seed transmission. Insects, contaminated water, and agricultural tools have also been implicated in viral spread. Symptomatology: PepMV infections can range from asymptomatic to severe. Common symptoms in tomato plants include leaf blistering, chlorosis, mosaic patterns, and fruit bleaching, flaming, and marbling. Severe cases may exhibit necrosis in leaves, stems, and fruits. Symptom severity depends on the viral isolate, tomato cultivar, timing of infection, and environmental conditions. Control: No PepMV‐resistant tomato varieties are currently available commercially. Therefore, preventive measures are commonly used strategies against PepMV. Cross‐protection has proven highly effective and is widely employed for disease management.

In Arabidopsis, receptor-like kinase BRASSINOSTEROID INSENSITIVE1-ASSOCIATED RECEPTOR KINASE 1 (BAK1) - INTERACTING RECEPTOR-LIKE KINASE 1 (BIR1) is a negative regulator of plant immunity and cell death. BIR1 was earlier described as a target of epigenetic and post-transcriptional silencing. Degradome analysis mapped predominant mRNA cleavage sites at the 5’-untranslated leader region (site A) and the protein-coding sequence (sites B and C). Here, we identified another cleavage site (D) within the BIR1 coding region and investigated the contribution of site-directed mRNA cleavage to BIR1 regulation. Mutations at B, C, and D sites enhanced mRNA stability by impairing transcript cleavage, resulting in increased BIR1 mRNA and protein accumulation. This regulation is disrupted in RNA silencing mutants, supporting a model of cis -directed small interfering RNA (siRNA)-mediated degradation. Cleavage events are highly localized, occurring at a limited number of sites that involve only a few siRNAs. Furthermore, our data reveal a repressive role for the 5’-leader in regulating BIR1 translation, potentially mediated by upstream open reading frames (uORFs) and a long non-coding RNA (lncRNA) derived from the natural antisense At4g39838 locus. This lncRNA is complementary to a TCA-rich repeat region encompassing cleavage site A, which may serve as a putative binding site. Together, these findings reveal a multilayered regulatory mechanism that integrates sRNA-mediated cleavage with translational control, with broader implications for the fine-tuning of stress-responsive gene expression during infection.

The family Alphaflexiviridae comprises plant- and fungus-infecting viruses with single-stranded, positive-sense RNA genomes ranging from 5.4 to 9 kb. Their virions are flexuous and filamentous, measuring 470–800 nm in length and 12–13 nm in diameter. The family includes 72 recognized species, classified into six genera: Allexivirus, Lolavirus, Platypuvirus, Potexvirus (plant-infecting), and Botrexvirus and Sclerodarnavirus (fungus-infecting). The genus Potexvirus is the largest, with 52 species, including Potexvirus ecspotati (potato virus X), an important crop pathogen and plant virology model. The genera are distinguished by genome organization and host range, while species differentiation relies on nucleotide and protein sequence identity thresholds. In this review, we summarize the current knowledge on the genomic structure, conserved genes, and phylogenetic relationships within Alphaflexiviridae, with a particular focus on the replicase and coat protein genes as signature markers. Additionally, we update the model of cellular remodeling driven by the triple gene block proteins, which are essential for virus movement, among other viral functions. Beyond their biological significance, alphaflexiviruses serve as valuable models for studying virus–host dynamics and hold potential applications in plant disease control and biotechnology. This review provides an updated framework for understanding Alphaflexiviridae and their broader impact on plant virology.

The family Botourmiaviridae includes viruses with a mono- or multi-segmented positive-sense RNA genome that infect plants and filamentous fungi. The family includes the genera Ourmiavirus (plant viruses), Botoulivirus, Betabotoulivirus, Magoulivirus, Scleroulivirus, Betascleroulivirus, Gammascleroulivirus, Deltascleroulivirus, Epsilonscleroulivirus, Penoulivirus, Rhizoulivirus and Betarhizoulivirus (fungal viruses). This summary is based on the International Committee on Taxonomy of Viruses (ICTV) Report on the family Botourmiaviridae, which is available at ictv.global/report/botourmiaviridae.

Botrytis species cause gray mold disease in more than 200 crops worldwide. To control this disease, chemical fungicides are usually applied. However, more sustainable control alternatives should be explored, such as the use of hypovirulent mycovirus-infected fungal strains. To determine the mycovirome of two Botrytis species, B. cinerea and B. prunorum, we reanalyzed RNA-Seq and small RNA-Seq data using different assembly programs and an updated viral database, aiming to identify new mycoviruses that were previously not described in the same dataset. New mycoviruses were identified, including those previously reported to infect or be associated with B. cinerea and Plasmopara viticola, such as Botrytis cinerea alpha-like virus 1 and Plasmopara viticola lesion-associated ourmia-like virus 80. Additionally, two novel narnaviruses, not previously identified infecting Botrytis species, have been characterized, tentatively named Botrytis cinerea narnavirus 1 and Botrytis narnavirus 1. The analysis of small RNAs suggested that all identified mycoviruses were targeted by the antiviral fungal mechanism, regardless of the viral genome type. In conclusion, the enlarged list of newly found viruses and the application of different bioinformatics approaches have enabled the identification of novel mycoviruses not previously described in Botrytis species, expanding the already extensive list.

Given the lack of specific studies on floral development in melon (Cucumis melo L.), we carried out an extensive study involving morphological and transcriptomic analyses to characterize floral development in this species. Using an andromonoecious line, we analysed the development of floral buds in male and hermaphrodite flowers with both light microscopy and scanning electron microscopy. Based on flower lengths, we established a correlation between the developmental stages and four main episodes of floral development and conducted an extensive RNA sequencing analysis of these episodes. We identified 12 stages of floral development, from the appearance of the floral meristems to anthesis. The main structural differences between male and hermaphrodite flowers appeared between stages 6 and 7; later stages of development leading to the formation of organs and structures in both types of flowers were also described. We analysed the gene expression patterns of the four episodes in flower development to find the genes that were specific to each given episode. Among others, we identified genes that defined the passage from one episode to the next according to the ABCDE model of floral development. This work combines a detailed morphological analysis and a comprehensive transcriptomic study to enable characterization of the structural and molecular mechanisms that determine the floral development of an andromonoecious genotype in melon. Taken together, our results provide a first insight into gene regulation networks in melon floral development that are crucial for flowering and pollen formation, highlighting potential targets for genetic manipulation to improve crop yield of melon in the future.

In this chapter, we describe a computational pipeline for the in silico detection of plant viruses by high-throughput sequencing (HTS) from total RNA samples. The pipeline is designed for the analysis of short reads generated using an Illumina platform and free-available software tools. First, we provide advice for high-quality total RNA purification, library preparation, and sequencing. The bioinformatics pipeline begins with the raw reads obtained from the sequencing machine and performs some curation steps to obtain long contigs. Contigs are blasted against a local database of reference nucleotide viral sequences to identify the viruses in the samples. Then, the search is refined by applying specific filters. We also provide the code to re-map the short reads against the viruses found to get information on sequencing depth and read coverage for each virus. No previous bioinformatics background is required, but basic knowledge of the Unix command line and R language is recommended. Key words Plant viruses Bioinformatics analysis High-throughput sequencing Short reads Contig assembly Virus detection