Viral Recognition and Evasion in Plants.

Viruses, causal agents of devastating diseases in plants, are obligate intracellular pathogens composed of a nucleic acid genome and a limited number of viral proteins. The diversity of plant viruses, their diminutive molecular nature, and their symplastic localization pose challenges to understanding the interplay between these pathogens and their hosts in the currently accepted framework of plant innate immunity. It is clear, nevertheless, that plants can recognize the presence of a virus and activate antiviral immune responses, although our knowledge of the breadth of invasion signals and the underpinning sensing events is far from complete. Below, I discuss some of the demonstrated or hypothesized mechanisms enabling viral recognition in plants, the step preceding the onset of antiviral immunity, as well as the strategies viruses have evolved to evade or suppress their detection.

Cell wall-mediated root development is targeted by a soil-borne bacterial pathogen to promote infection.

Plant pathogens manipulate host development, facilitating colonization and proliferation. Ralstonia solanacearum is a soil-borne bacterial pathogen that penetrates roots and colonizes plants through the vascular system, causing wilting and death. Here, we find that RipAC, an effector protein from R. solanacearum, alters root development in Arabidopsis, promoting the formation of lateral roots and root hairs. RipAC interacts with CELLULOSE SYNTHASE (CESA)-INTERACTIVE PROTEIN 1 (CSI1), which regulates the activity of CESA complexes at the plasma membrane. RipAC disrupts CESA-CSI1 interaction, leading to a reduction in cellulose content, root developmental alterations, and a promotion of bacterial pathogenicity. We find that CSI1 also associates with the receptor kinase FERONIA, forming a complex that negatively regulates immunity in roots; this interaction, however, is not affected by RipAC. Our work reveals a bacterial virulence strategy that selectively affects the activities of a host target, promoting anatomical alterations that facilitate infection without causing activation of immunity.

Youth Are the Experts! Youth Participatory Action Research to Address the Adolescent Mental Health Crisis.

Adolescent mental health is an urgent global public health issue. Youth participatory action research is an effective strategy to amplify youth voices and can serve as a catalyst for evidence-based action addressing the mental health crisis. To illustrate the benefits of youth participatory action research for informing community health, we describe an ongoing collaboration with a youth council located in the central coast of California, USA. Research methods included an anonymous online self-report survey to gather information about the mental health of high school students in 2020 (n = 176) and 2022 (n = 234), 93% Latinx/Mexican American. Both surveys included a four-item patient health questionnaire to screen for depression and anxiety risk, in addition to scaled and open-ended survey questions selected by the youth leaders based on their research questions. Quantitative and qualitative results indicated a significant but small decrease in mental health risk, and a continued need for resources to access mental health support. Results led to community-based action aimed at improving local youth mental health. The interdisciplinary research team (psychology and public health) and youth leaders share reflections highlighting the innovative, empowering, and transformative impact of youth participatory action research as a tool for improving community health.

The Ti-TAN plasmid toolbox for TurboID-based proximity labeling assays in Nicotiana benthamiana.

The Ti-TAN TurboID plasmid toolbox enables proximity labeling applications in transient assays in Nicotiana benthamiana in a fast and cost-efficient manner, making TurboID-based proximity labeling broadly accessible to plant scientists.

The m6A reader ECT1 drives mRNA sequestration to dampen salicylic acid-dependent stress responses in Arabidopsis.

N 6-methyladenosine (m6A) is a common epitranscriptional mRNA modification in eukaryotes. Thirteen putative m6A readers, mostly annotated as EVOLUTIONARILY CONSERVED C-TERMINAL REGION (ECT) proteins, have been identified in Arabidopsis (Arabidopsis thaliana), but few have been characterized. Here, we show that the Arabidopsis m6A reader ECT1 modulates salicylic acid (SA)-mediated plant stress responses. ECT1 undergoes liquid-liquid phase separation in vitro, and its N-terminal prion-like domain is critical for forming in vivo cytosolic biomolecular condensates in response to SA or bacterial pathogens. Fluorescence-activated particle sorting coupled with quantitative PCR analyses unveiled that ECT1 sequesters SA-induced m6A modification-prone mRNAs through its conserved aromatic cage to facilitate their decay in cytosolic condensates, thereby dampening SA-mediated stress responses. Consistent with this finding, ECT1 overexpression promotes bacterial multiplication in plants. Collectively, our findings unequivocally link ECT1-associated cytosolic condensates to SA-dependent plant stress responses, advancing the current understanding of m6A readers and the SA signaling network.

From Metagenomics to Ecogenomics: NGS-Based Approaches for Discovery of New Circular DNA Single-Stranded Viral Species.

Viruses comprise the most abundant genetic material in the biosphere; however, global viral genomic population (virome) has been largely underestimated. Recently, high-throughput sequencing (HTS) has provided a powerful tool for the detection of known viruses and the discovery of novel viral species from environmental and individual samples using metagenomics and ecogenomics approaches, respectively. Viruses with circular DNA single-stranded (ssDNA) genomes belonging to the begomovirus genera (family Geminiviridae) constitute the largest group of emerging plant viruses worldwide. The knowledge of begomoviruses viromes is mostly restricted to crop plant systems; nevertheless, it has been described that noncultivated plants specifically at the interface between wild and cultivated plants are important reservoirs leading to viral evolution and the emergence of new diseases. Here we present a protocol that allows the identification and isolation of known and novel begomoviruses species infecting cultivated and noncultivated plant species. The method consists of circular viral molecules enrichment by rolling circle amplification (RCA) from begomovirus-positive total plant DNA, followed by NGS-based metagenomic sequencing. Subsequently, metagenomic reads are processed for taxonomic classification using Viromescan software and a customized Geminiviridae family database, and begomovirus-related reads are used for contigs assembly and annotation using Spades software and Blastn algorithm, respectively. Then, the obtained begomovirus-related signatures are used as templates for specific primers design and implemented for PCR-based ecogenomic identification of individual samples harboring the corresponding viral species. Lastly, full-length begomovirus genomes are obtained by RCA-based amplification from total plant DNA of selected individual samples, cloning, and viral molecular identity corroborated by Sanger sequencing. Conclusively, the identification and isolation of a novel monopartite begomovirus species native to the New World (NW) named Gallium leaf deformation virus (GLDV) is shown.

Two viral proteins translated from one open reading frame target different layers of plant defense.

Multilayered defense responses are activated upon pathogen attack. Viruses utilize a number of strategies to maximize the coding capacity of their small genomes and produce viral proteins for infection, including suppression of host defense. Here, we reveal translation leakage as one of these strategies: two viral effectors encoded by tomato golden mosaic virus, chloroplast-localized C4 (cC4) and membrane-associated C4 (mC4), are translated from two in-frame start codons and function cooperatively to suppress defense. cC4 localizes in chloroplasts, to which it recruits NbPUB4 to induce ubiquitination of the outer membrane; as a result, this organelle is degraded, and chloroplast-mediated defenses are abrogated. However, chloroplast-localized cC4 induces the production of singlet oxygen (1O2), which in turn promotes translocation of the 1O2 sensor NbMBS1 from the cytosol to the nucleus, where it activates expression of the CERK1 gene. Importantly, an antiviral effect exerted by CERK1 is countered by mC4, localized at the plasma membrane. mC4, like cC4, recruits NbPUB4 and promotes the ubiquitination and subsequent degradation of CERK1, suppressing membrane-based, receptor-like kinase-dependent defenses. Importantly, this translation leakage strategy seems to be conserved in multiple viral species and is related to host range. This finding suggests that stacking of different cellular antiviral responses could be an effective way to abrogate viral infection and engineer sustainable resistance to major crop viral diseases in the field.

Plant and animal positive-sense single-stranded RNA viruses encode small proteins important for viral infection in their negative-sense strand.

Positive-sense single-stranded RNA (+ssRNA) viruses, the most abundant viruses of eukaryotes in nature, require the synthesis of negative-sense RNA (-RNA) using their genomic (positive-sense) RNA (+RNA) as a template for replication. Based on current evidence, viral proteins are translated via viral +RNAs, whereas -RNA is considered to be a viral replication intermediate without coding capacity. Here, we report that plant and animal +ssRNA viruses contain small open reading frames (ORFs) in their -RNA (reverse ORFs [rORFs]). Using turnip mosaic virus (TuMV) as a model for plant +ssRNA viruses, we demonstrate that small proteins encoded by rORFs display specific subcellular localizations, and confirm the presence of rORF2 in infected cells through mass spectrometry analysis. The protein encoded by TuMV rORF2 forms punctuate granules that are localized in the perinuclear region and co-localized with viral replication complexes. The rORF2 protein can directly interact with the viral RNA-dependent RNA polymerase, and mutation of rORF2 completely abolishes virus infection, whereas ectopic expression of rORF2 rescues the mutant virus. Furthermore, we show that several rORFs in the -RNA of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have the ability to suppress type I interferon production and facilitate the infection of vesicular stomatitis virus. In addition, we provide evidence that TuMV might utilize internal ribosome entry sites to translate these small rORFs. Taken together, these findings indicate that the -RNA of +ssRNA viruses can also have the coding capacity and that small proteins encoded therein play critical roles in viral infection, revealing a viral proteome larger than previously thought.

Changes in the Acetylcholinesterase Enzymatic Activity in Tumor Development and Progression.

Acetylcholinesterase is a well-known protein because of the relevance of its enzymatic activity in the hydrolysis of acetylcholine in nerve transmission. In addition to the catalytic action, it exerts non-catalytic functions; one is associated with apoptosis, in which acetylcholinesterase could significantly impact the survival and aggressiveness observed in cancer. The participation of AChE as part of the apoptosome could explain the role in tumors, since a lower AChE content would increase cell survival due to poor apoptosome assembly. Likewise, the high Ach content caused by the reduction in enzymatic activity could induce cell survival mediated by the overactivation of acetylcholine receptors (AChR) that activate anti-apoptotic pathways. On the other hand, in tumors in which high enzymatic activity has been observed, AChE could be playing a different role in the aggressiveness of cancer; in this review, we propose that AChE could have a pro-inflammatory role, since the high enzyme content would cause a decrease in ACh, which has also been shown to have anti-inflammatory properties, as discussed in this review. In this review, we analyze the changes that the enzyme could display in different tumors and consider the different levels of regulation that the acetylcholinesterase undergoes in the control of epigenetic changes in the mRNA expression and changes in the enzymatic activity and its molecular forms. We focused on explaining the relationship between acetylcholinesterase expression and its activity in the biology of various tumors. We present up-to-date knowledge regarding this fascinating enzyme that is positioned as a remarkable target for cancer treatment.

More than annealing: RNAi is not alone in the fight against plant viruses.

Cellular organisms have evolved different strategies to defend themselves against the invasion by viruses. In plants, RNA interference (RNAi) or RNA silencing, which is triggered by virus-derived double-stranded (ds)RNA, is considered the main antiviral defence mechanism. Martínez-Pérez et al have now uncovered an additional plant antiviral pathway, termed by the authors "m6 A-YTHDF axis," which relies on the modification and subsequent recognition of the viral RNA.

Geminivirus C5 proteins mediate formation of virus complexes at plasmodesmata for viral intercellular movement.

Movement proteins (MPs) encoded by plant viruses deliver viral genomes to plasmodesmata (PD) to ensure intracellular and intercellular transport. However, how the MPs encoded by monopartite geminiviruses are targeted to PD is obscure. Here, we demonstrate that the C5 protein of tomato yellow leaf curl virus (TYLCV) anchors to PD during the viral infection following trafficking from the nucleus along microfilaments in Nicotiana benthamiana. C5 could move between cells and partially complement the traffic of a movement-deficient turnip mosaic virus (TuMV) mutant (TuMV-GFP-P3N-PIPO-m1) into adjacent cells. The TYLCV-C5 null mutant (TYLCV-mC5) attenuates viral pathogenicity and decreases viral DNA and protein accumulation, and ectopic overexpression of C5 enhances viral DNA accumulation. Interaction assays between TYLCV-C5 and the other eight viral proteins described in TYLCV reveal that C5 associates with C2 in the nucleus and with V2 in the cytoplasm and at PD. The V2 protein is mainly localized in the nucleus and cytoplasmic granules when expressed alone; in contrast, V2 forms small punctate granules at PD when co-expressed with C5 or in TYLCV-infected cells. The interaction of V2 and C5 also facilitates their nuclear export. Furthermore, C5-mediated PD localization of V2 is conserved in two other geminiviruses. Therefore, this study solves a long-sought-after functional connection between PD and the geminivirus movement and improves our understanding of geminivirus-encoded MPs and their potential cellular and molecular mechanisms.

Functional identification of a novel C7 protein of tomato yellow leaf curl virus.

Tomato yellow leaf curl virus (TYLCV) is a monopartite geminivirus, and one of the most devastating plant viruses in the world. TYLCV is traditionally known to encode six viral proteins in bidirectional and partially overlapping open reading frames (ORFs). However, recent studies have shown that TYLCV encodes additional small proteins with specific subcellular localizations and potential virulence functions. Here, a novel protein named C7, encoded by a newly-described ORF in the complementary strand, was identified as part of the TYLCV proteome using mass spectrometry. The C7 protein localized to the nucleus and cytoplasm, both in the absence and presence of the virus. C7 was found to interact with two other TYLCV-encoded proteins: with C2 in the nucleus, and with V2 in the cytoplasm, forming conspicuous granules. Mutation of C7 start codon ATG to ACG to block the translation of C7 delayed the onset of viral infection, and the mutant virus caused milder virus symptoms and less accumulations of viral DNAs and proteins. Using the potato virus X (PVX)-based recombinant vector, we found that ectopic overexpression of C7 resulted in more severe mosaic symptoms and promoted a higher accumulation of PVX-encoded coat protein in the late virus infection stage. In addition, C7 was also found to inhibit GFP-induced RNA silencing moderately. This study demonstrates that the novel C7 protein encoded by TYLCV is a pathogenicity factor and a weak RNA silencing suppressor, and that it plays a critical role during TYLCV infection.

Hierarchical regulatory module GENOMES UNCOUPLED1-GOLDEN2-LIKE1/2-WRKY18/40 modulates salicylic acid signaling.

Chloroplast-to-nucleus retrograde signaling (RS) pathways are critical in modulating plant development and stress adaptation. Among chloroplast proteins mediating RS pathways, GENOMES UNCOUPLED1 (GUN1) represses the transcription of the nuclear transcription factors GOLDEN2-LIKE1 (GLK1) and GLK2 that positively regulate chloroplast biogenesis. Given the extensive exploration of the function of GUN1 in biogenic RS carried out in previous years, our understanding of its role in plant stress responses remains scarce. Here, we revealed that GUN1 contributes to the expression of salicylic acid (SA)-responsive genes (SARGs) through transcriptional repression of GLK1/2 in Arabidopsis (Arabidopsis thaliana). Loss of GUN1 significantly compromised the SA responsiveness in plants, concomitant with the upregulation of GLK1/2 transcripts. In contrast, knockout of GLK1/2 potentiated the expression of SARGs and led to enhanced stress responses. Chromatin immunoprecipitation, coupled with quantitative PCR and related reverse genetic approaches, unveiled that in gun1, GLK1/2 might modulate SA-triggered stress responses by stimulating the expression of WRKY18 and WRKY40, transcriptional repressors of SARGs. In summary, we demonstrate that a hierarchical regulatory module, consisting of GUN1-GLK1/2-WRKY18/40, modulates SA signaling, opening a research avenue regarding a latent GUN1 function in plant-environment interactions.

The transcriptional regulator JAZ8 interacts with the C2 protein from geminiviruses and limits the geminiviral infection in Arabidopsis.

Jasmonates (JAs) are phytohormones that finely regulate critical biological processes, including plant development and defense. JASMONATE ZIM-DOMAIN (JAZ) proteins are crucial transcriptional regulators that keep JA-responsive genes in a repressed state. In the presence of JA-Ile, JAZ repressors are ubiquitinated and targeted for degradation by the ubiquitin/proteasome system, allowing the activation of downstream transcription factors and, consequently, the induction of JA-responsive genes. A growing body of evidence has shown that JA signaling is crucial in defending against plant viruses and their insect vectors. Here, we describe the interaction of C2 proteins from two tomato-infecting geminiviruses from the genus Begomovirus, tomato yellow leaf curl virus (TYLCV) and tomato yellow curl Sardinia virus (TYLCSaV), with the transcriptional repressor JAZ8 from Arabidopsis thaliana and its closest orthologue in tomato, SlJAZ9. Both JAZ and C2 proteins colocalize in the nucleus, forming discrete nuclear speckles. Overexpression of JAZ8 did not lead to altered responses to TYLCV infection in Arabidopsis; however, knock-down of JAZ8 favors geminiviral infection. Low levels of JAZ8 likely affect the viral infection specifically, since JAZ8-silenced plants neither display obvious developmental phenotypes nor present differences in their interaction with the viral insect vector. In summary, our results show that the geminivirus-encoded C2 interacts with JAZ8 in the nucleus, and suggest that this plant protein exerts an anti-geminiviral effect.

Manipulation of plant RNA biology by geminiviruses.

Viruses are intracellular parasites that have evolved to effectively manipulate the cells they infect. As a result of the viral infection, multiple cellular processes are altered, suppressed, or redirected, partially due to the viral co-option of the host's molecular machinery. RNA biology plays a central role in virus-host interactions, since it is at the basis of viral gene expression, splicing of viral transcripts, anti-viral RNA silencing, and-at least in the case of RNA viruses-genome replication, and therefore is heavily targeted by viruses. The plant DNA geminiviruses, causal agents of devasting diseases in crops worldwide, are no exception, and RNA processing is tightly entrenched in their infection cycle. In this review, we will discuss the relevance of the manipulation of RNA biology by geminiviruses for a successful viral infection and the underlying molecular mechanisms, and suggest some of the multiple remaining open questions in this field.

The primase subunits of DNA polymerase α, PRIM1 and PRIM2, are required for the replication of the geminivirus tomato yellow leaf curl virus in the host plant.

Geminiviruses are causal agents of devastating diseases in crops. Geminiviral genomes are single-stranded (ss) circular DNA molecules that replicate in the nucleus of the infected cell through double-stranded (ds) intermediates by co-opting the plant DNA replication machinery. However, the identity of the plant DNA polymerases enabling geminiviral replication has remained largely elusive. Recently, we showed that DNA polymerase α mediates the ss-to-ds conversion of tomato yellow leaf curl virus (TYLCV), and is therefore essential for the infection. Here, we provide data indicating that the primase subunits of DNA polymerase α, PRIM1 and PRIM2, are also required for TYLCV replication.

Percepción en las ocupaciones de mujeres con cáncer de mama que ejercen rol de madre trabajadora: una mirada desde el Modelo Persona Ambiente Ocupación / Percepção das ocupações de mulheres com câncer de mama que exercem o papel de mãe trabalhadora: um olhar a partir do Modelo Pessoa Ambiente Ocupação / Perception of the occupational roles of working mothers with breast cancer: a view from the Person-Environment-Occupation Model

Resumen En esta investigación se aborda cómo las mujeres que cumplen el rol de madre trabajadora perciben el impacto en estos roles posterior a la experiencia de tener/haber tenido cáncer de mama, desde el Modelo Persona Ambiente Ocupación, considerando todos sus componentes y como su interacción influye en el desempeño ocupacional. La metodología utilizada se centra en la recopilación de información, por medio de entrevistas realizadas a 10 mujeres acerca de sus experiencias en relación con este diagnóstico, de las cuales 5 están en tratamiento, y las restantes se encuentran recuperadas. De acuerdo con los resultados, las mujeres perciben que su desempeño se vio impactado, debido a las consecuencias físicas, socioemocionales y cognitivas que conlleva el tener/haber tenido cáncer de mama. A su vez se generó un impacto en los roles que desempeñan estas mujeres, por lo cual se evidenció la importancia de contar con redes de apoyo durante todo el proceso. Debido a esto, es necesario llevar a cabo futuras investigaciones que puedan contribuir y fundamentar la importancia de la terapia ocupacional en esta patología para así favorecer un tratamiento integral, considerando sus ocupaciones significativas y los contextos en los cuales se desenvuelven.

Resumo Esta pesquisa aborda, a partir do Modelo Pessoa Ambiente Ocupação, como as mulheres que cumprem o papel de mães trabalhadoras percebem o impacto destes papéis, após a experiência de ter/ter tido câncer de mama, considerando todos os seus componentes e como sua interação influencia o desempenho ocupacional. A metodologia utilizada centra-se na coleta de informação, através de entrevistas a 10 mulheres sobre as suas vivências em relação a este diagnóstico, das quais cinco estão em tratamento e as restantes recuperadas. De acordo com os resultados, as mulheres percebem que seu desempenho foi impactado, devido às consequências físicas, socioemocionais e cognitivas de ter/ter tido câncer de mama. Ao mesmo tempo, gerou-se um impacto nos papéis que essas mulheres desempenham, para o qual ficou evidente a importância de contar com redes de apoio ao longo do processo. Diante disso, faz-se necessária a realização de pesquisas futuras que possam contribuir e fundamentar a importância da terapia ocupacional nessa patologia de forma a favorecer um tratamento integral, considerando suas ocupações significativas e os contextos em que estão inseridas.

Abstract This research addresses the perceptions of women who identify as working mothers regarding the impact on their occupational roles following a breast cancer diagnosis, based on the Person-Environment-Occupation Model. The methodology involved conducting interviews with ten women about their experiences related to their breast cancer diagnosis, of which five were undergoing treatment and five had recovered. The results reveal that these women perceive their performance to be impacted by the physical, socioemotional, and cognitive consequences of having or having had breast cancer. Furthermore, the illness also affected the various roles they inhabited, highlighting the importance of support networks throughout the process. Therefore, future research is essential to underscore the importance of occupational therapy in treating this condition, considering the women's significant occupations and the contexts in which they occur.

The V2 Protein from the Geminivirus Tomato Yellow Leaf Curl Virus Largely Associates to the Endoplasmic Reticulum and Promotes the Accumulation of the Viral C4 Protein in a Silencing Suppression-Independent Manner.

Viruses are strict intracellular parasites that rely on the proteins encoded in their genomes for the effective manipulation of the infected cell that ultimately enables a successful infection. Viral proteins have to be produced during the cell invasion and takeover in sufficient amounts and in a timely manner. Silencing suppressor proteins evolved by plant viruses can boost the production of viral proteins; although, additional mechanisms for the regulation of viral protein production likely exist. The strongest silencing suppressor encoded by the geminivirus tomato yellow leaf curl virus (TYLCV) is V2: V2 suppresses both post-transcriptional and transcriptional gene silencing (PTGS and TGS), activities that are associated with its localization in punctate cytoplasmic structures and in the nucleus, respectively. However, V2 has been previously described to largely localize in the endoplasmic reticulum (ER), although the biological relevance of this distribution remains mysterious. Here, we confirm the association of V2 to the ER in Nicotiana benthamiana and assess the silencing suppression activity-independent impact of V2 on protein accumulation. Our results indicate that V2 has no obvious influence on the localization of ER-synthesized receptor-like kinases (RLKs) or ER quality control (ERQC)/ER-associated degradation (ERAD), but dramatically enhances the accumulation of the viral C4 protein, which is co-translationally myristoylated, possibly in proximity to the ER. By using the previously described V2C84S/86S mutant, in which the silencing suppression activity is abolished, we uncouple RNA silencing from the observed effect. Therefore, this work uncovers a novel function of V2, independent of its capacity to suppress silencing, in the promotion of the accumulation of another crucial viral protein.