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.

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.

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.

A fungal protease named AsES triggers antiviral immune responses and effectively restricts virus infection in arabidopsis and Nicotiana benthamiana plants.

BACKGROUND AND AIMS: Plants have evolved complex mechanisms to fight against pathogens. Among these mechanisms, pattern-triggered immunity (PTI) relies on the recognition of conserved microbe- or pathogen-associated molecular patterns (MAMPs or PAMPs, respectively) by membrane-bound receptors. Indeed, PTI restricts virus infection in plants and, in addition, BRI1-associated kinase 1 (BAK1), a central regulator of PTI, plays a role in antiviral resistance. However, the compounds that trigger antiviral defences, along with their molecular mechanisms of action, remain mostly elusive. Herein, we explore the role of a fungal extracellular subtilase named AsES in its capacity to trigger antiviral responses. METHODS: In this study, we obtained AsES by recombinant expression, and evaluated and characterized its capacity to trigger antiviral responses against Tobacco mosaic virus (TMV) by performing time course experiments, analysing gene expression, virus movement and callose deposition. KEY RESULTS: The results of this study provide direct evidence that exogenous treatment with recombinant AsES increases a state of resistance against TMV infection, in both arabidopsis and Nicotiana benthamiana plants. Also, the antiviral PTI response exhibited by AsES in arabidopsis is mediated by the BAK1/SERK3 and BKK1/SERK4 co-receptors. Moreover, AsES requires a fully active salicylic acid (SA) signalling pathway to restrict the TMV movement by inducing callose deposition. Additionally, treatment with PSP1, a biostimulant based on AsES as the active compound, showed an increased resistance against TMV in N. benthamiana and tobacco plants. CONCLUSIONS: AsES is a fungal serine protease which triggers antiviral responses relying on a conserved mechanism by means of the SA signalling pathway and could be exploited as an effective and sustainable biotechnology strategy for viral disease management in plants.

TuMV triggers stomatal closure but reduces drought tolerance in Arabidopsis.

Compatible plant viral infections are a common cause of agricultural losses worldwide. Characterization of the physiological responses controlling plant water management under combined stresses is of great interest in the current climate change scenario. We studied the outcome of TuMV infection on stomatal closure and water balance, hormonal balance and drought tolerance in Arabidopsis. TuMV infection reduced stomatal aperture concomitantly with diminished gas exchange rate, daily water consumption and rosette initial dehydration rate. Infected plants overaccumulated salicylic acid and abscisic acid and showed altered expression levels of key ABA homeostasis genes including biosynthesis and catabolism. Also the expression of ABA signalling gene ABI2 was induced and ABCG40 (which imports ABA into guard cells) was highly induced upon infection. Hypermorfic abi2-1 mutant plants, but no other ABA or SA biosynthetic, signalling or degradation mutants tested abolished both stomatal closure and low stomatal conductance phenotypes caused by TuMV. Notwithstanding lower relative water loss during infection, plants simultaneously subjected to drought and viral stresses showed higher mortality rates than mock-inoculated drought stressed controls, alongside downregulation of drought-responsive gene RD29A. Our findings indicate that despite stomatal closure triggered by TuMV, additional phenomena diminish drought tolerance upon infection.

Multiomics analyses reveal the roles of the ASR1 transcription factor in tomato fruits.

The transcription factor ASR1 (ABA, STRESS, RIPENING 1) plays multiple roles in plant responses to abiotic stresses as well as being involved in the regulation of central metabolism in several plant species. However, despite the high expression of ASR1 in tomato fruits, large scale analyses to uncover its function in fruits are still lacking. In order to study its function in the context of fruit ripening, we performed a multiomics analysis of ASR1-antisense transgenic tomato fruits at the transcriptome and metabolome levels. Our results indicate that ASR1 is involved in several pathways implicated in the fruit ripening process, including cell wall, amino acid, and carotenoid metabolism, as well as abiotic stress pathways. Moreover, we found that ASR1-antisense fruits are more susceptible to the infection by the necrotrophic fungus Botrytis cinerea. Given that ASR1 could be regulated by fruit ripening regulators such as FRUITFULL1/FRUITFULL2 (FUL1/FUL2), NON-RIPENING (NOR), and COLORLESS NON-RIPENING (CNR), we positioned it in the regulatory cascade of red ripe tomato fruits. These data extend the known range of functions of ASR1 as an important auxiliary regulator of tomato fruit ripening.

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.

Single-stranded oligodeoxynucleotides induce plant defence in Arabidopsis thaliana.

BACKGROUND AND AIMS: Single-stranded DNA oligodeoxynucleotides (ssODNs) have been shown to elicit immune responses in mammals. In plants, RNA and genomic DNA can activate immunity, although the exact mechanism through which they are sensed is not clear. The aim of this work was to study the possible effect of ssODNs on plant immunity. KEY RESULTS: The ssODNs IMT504 and 2006 increased protection against the pathogens Pseudomonas syringae pv. tomato DC3000 and Botrytis cinerea but not against tobacco mosaic virus-Cg when infiltrated in Arabidopsis thaliana. In addition, ssODNs inhibited root growth and promoted stomatal closure in a concentration-dependent manner, with half-maximal effective concentrations between 0.79 and 2.06 µm. Promotion of stomatal closure by ssODNs was reduced by DNase I treatment. It was also diminished by the NADPH oxidase inhibitor diphenyleneiodonium and by coronatine, a bacterial toxin that inhibits NADPH oxidase-dependent reactive oxygen species (ROS) synthesis in guard cells. In addition it was found that ssODN-mediated stomatal closure was impaired in bak1-5, bak1-5/bkk1, mpk3 and npr1-3 mutants. ssODNs also induced early expression of MPK3, WRKY33, PROPEP1 and FRK1 genes involved in plant defence, an effect that was reduced in bak1-5 and bak1-5/bkk1 mutants. CONCLUSIONS: ssODNs are capable of inducing protection against pathogens through the activation of defence genes and promotion of stomatal closure through a mechanism similar to that of other elicitors of plant immunity, which involves the BAK1 co-receptor, and ROS synthesis.

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.

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.

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.

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.

Three-year study of DNA cytosine methylation dynamics in transplanted Malbec grapevines.

DNA cytosine methylation, an epigenetic mechanism involved in gene regulation and genome stability, remains poorly understood in terms of its role under changing environmental conditions. Previous research using methylation-sensitive amplified polymorphism (MSAP) markers in a Vitis vinifera L. cv. Malbec clone showed vineyard-specific DNA methylation polymorphism, but no change in overall methylation levels. To complement these findings, the present study investigates the intra-seasonal epigenetic dynamics between genetically identical plants grown in different vineyards through a transplanting experiment. Cuttings of the same clone, showing differential methylation patterns imposed by the vineyard of origin (Agrelo and Gualtallary), were cultivated in a common vineyard (Lunlunta). Using high-performance liquid chromatography-ultraviolet detection, the quantification of global DNA 5-methylcytosine (5-mC) levels revealed relatively low overall 5-mC percentages in grapevines, with higher levels in Agrelo (5.8%) compared to Gualtallary plants (3.7%). The transplanted plants maintained the 5-mC levels differences between vineyards (9.8% vs 6.2%), which equalized in subsequent seasons (7.5% vs 7%). Additionally, the study examined 5-mC polymorphism using MSAP markers in Lunlunta transplanted plants over three seasons. The observed differences between vineyards in MSAP patterns during the initial growing season gradually diminished, suggesting a reprogramming of the hemimethylated pattern following implantation in the common vineyard. In contrast, the non-methylated pattern exhibited greater stability, indicating a potential memory effect. Overall, this study provides valuable insights into the dynamic nature of DNA methylation in grapevines under changing environmental conditions, with potential implications for crop management and breeding strategies.

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.

Coupling virus-induced gene silencing to exogenous green fluorescence protein expression provides a highly efficient system for functional genomics in Arabidopsis and across all stages of tomato fruit development.

Since the advent of the postgenomic era, efforts have focused on the development of rapid strategies for annotating plant genes of unknown function. Given its simplicity and rapidity, virus-induced gene silencing (VIGS) has become one of the preeminent approaches for functional analyses. However, several problems remain intrinsic to the use of such a strategy in the study of both metabolic and developmental processes. The most prominent of these is the commonly observed phenomenon of "sectoring" the tissue regions that are not effectively targeted by VIGS. To better discriminate these sectors, an effective marker system displaying minimal secondary effects is a prerequisite. Utilizing a VIGS system based on the tobacco rattle virus vector, we here studied the effect of silencing the endogenous phytoene desaturase gene (pds) and the expression and subsequent silencing of the exogenous green fluorescence protein (gfp) on the metabolism of Arabidopsis (Arabidopsis thaliana) leaves and tomato (Solanum lycopersicum) fruits. In leaves, we observed dramatic effects on primary carbon and pigment metabolism associated with the photobleached phenotype following the silencing of the endogenous pds gene. However, relatively few pleiotropic effects on carbon metabolism were observed in tomato fruits when pds expression was inhibited. VIGS coupled to gfp constitutive expression revealed no significant metabolic alterations after triggering of silencing in Arabidopsis leaves and a mild effect in mature green tomato fruits. By contrast, a wider impact on metabolism was observed in ripe fruits. Silencing experiments with an endogenous target gene of interest clearly demonstrated the feasibility of cosilencing in this system; however, carefully constructed control experiments are a prerequisite to prevent erroneous interpretation.

Changes in micro RNA expression in a wild tuber-bearing Solanum species induced by 5-Azacytidine treatment.

Phenotypic plasticity is often postulated as a principal characteristic of tuber-bearing wild Solanum species. The hypotheses to explore this observation have been developed based on the presence of genetic variation. In this context, evolutionary changes and adaptation are impossible without genetic variation. However, epigenetic effects, which include DNA methylation and microRNAs expression control, could be another source of phenotypic variation in ecologically relevant traits. To achieve a detailed mechanistic understanding of these processes, it is necessary to separate epigenetic from DNA sequence-based effects and to evaluate their relative importance on phenotypic variability. We explored the potential relevance of epigenetic effects in individuals with the same genotype. For this purpose, a clone of the wild potato Solanum ruiz-lealii, a non-model species in which natural methylation variability has been demonstrated, was selected and its DNA methylation was manipulated applying 5-Azacytidine (AzaC), a demethylating agent. The AzaC treatment induced early flowering and changes in leaf morphology. Using quantitative real-time PCR, we identified four miRNAs up-regulated in the AzaC-treated plants. One of them, miRNA172, could play a role on the early flowering phenotype. In this work, we showed that the treatment with AzaC could provide meaningful results allowing to study both the phenotypic plasticity in tuber-bearing Solanum species and the inter-relation between DNA methylation and miRNA accumulations in a wide range of species.

Unraveling the Complexity of the Rhomboid Serine Protease 4 Family of Babesia bovis Using Bioinformatics and Experimental Studies.

Babesia bovis, a tick-transmitted apicomplexan protozoon, infects cattle in tropical and subtropical regions around the world. In the apicomplexans Toxoplasma gondii and Plasmodium falciparum, rhomboid serine protease 4 (ROM4) fulfills an essential role in host cell invasion. We thus investigated B. bovis ROM4 coding genes; their genomic organization; their expression in in vitro cultured asexual (AS) and sexual stages (SS); and strain polymorphisms. B. bovis contains five rom4 paralogous genes in chromosome 2, which we have named rom4.1, 4.2, 4.3, 4.4 and 4.5. There are moderate degrees of sequence identity between them, except for rom4.3 and 4.4, which are almost identical. RT-qPCR analysis showed that rom4.1 and rom4.3/4.4, respectively, display 18-fold and 218-fold significantly higher (p < 0.01) levels of transcription in SS than in AS, suggesting a role in gametogenesis-related processes. In contrast, transcription of rom4.4 and 4.5 differed non-significantly between the stages. ROM4 polymorphisms among geographic isolates were essentially restricted to the number of tandem repeats of a 29-amino acid sequence in ROM4.5. This sequence repeat is highly conserved and predicted as antigenic. B. bovis ROMs likely participate in relevant host−pathogen interactions and are possibly useful targets for the development of new control strategies against this pathogen.

Current recommendations and novel strategies for sustainable management of soybean sudden death syndrome.

The increase in food production requires reduction of the damage caused by plant pathogens, minimizing the environmental impact of management practices. Soil-borne pathogens are among the most relevant pathogens that affect soybean crop yield. Soybean sudden death syndrome (SDS), caused by several distinct species of Fusarium, produces significant yield losses in the leading soybean-producing countries in North and South America. Current management strategies for SDS are scarce since there are no highly resistant cultivars and only a few fungicide seed treatments are available. Because of this, innovative approaches for SDS management need to be developed. Here, we summarize recently explored strategies based on plant nutrition, biological control, priming of plant defenses, host-induced gene silencing, and the development of new SDS-resistance cultivars using precision breeding techniques. Finally, sustainable management of SDS should also consider cultural control practices with minimal environmental impact. © 2021 Society of Chemical Industry.

Antiviral efficacy of short-hairpin RNAs and artificial microRNAs targeting foot-and-mouth disease virus.

RNA interference (RNAi) is a well-conserved mechanism in eukaryotic cells that directs post-transcriptional gene silencing through small RNA molecules. RNAi has been proposed as an alternative approach for rapid and specific control of viruses including foot-and-mouth disease virus (FMDV), the causative agent of a devastating animal disease with high economic impact. The aim of this work was to assess the antiviral activity of different small RNA shuttles targeting the FMDV RNA-dependent RNA polymerase coding sequence (3D). Three target sequences were predicted within 3D considering RNA accessibility as a major criterion. The silencing efficacy of short-hairpin RNAs (shRNAs) and artificial microRNAs (amiRNAs) targeting the selected sequences was confirmed in fluorescent reporter assays. Furthermore, BHK-21 cells transiently expressing shRNAs or amiRNAs proved 70 to >95% inhibition of FMDV growth. Interestingly, dual expression of amiRNAs did not improve FMDV silencing. Lastly, stable cell lines constitutively expressing amiRNAs were established and characterized in terms of antiviral activity against FMDV. As expected, viral replication in these cell lines was delayed. These results show that the target RNA-accessibility-guided approach for RNAi design rendered efficient amiRNAs that constrain FMDV replication. The application of amiRNAs to complement FMDV vaccination in specific epidemiological scenarios shall be explored further.