Plant Molecular Biology

Activation domains are used as critical components of artificial gene modification tools for genetic breeding. The high efficiency of the activation domain relies on the host plant. However, no activation domain has been identified that originates from Chinese fir (Cunninghamia lanceolate). In this study, a novel strong activator was identified from the whole Chinese fir cDNA library. This plant conserved activator was named TAC 3 (Transcriptional Activation domain from Chinese fir 3). C-terminal 70 amino acids of TAC (TAC3d) have a stronger ability than the commonly used strong activation domain of the virus protein VP16, or the strong plant activation domain, EDLL, in Chinese fir. Through Dual-luciferase assay, phenomic analysis and FT (Flowering Locus T [FT]) quantification, it was shown that, TAC3d can overcome the transcriptional repression of strong plant repressors (Flowering Locus C [FLC]) when fused to its C-terminal domain, thus inhibit the repression of FT expression. In conclusion, for the first time, an activation domain has been identified from Chinese fir. TAC3, which can be used for precise gene activation in Chinese fir in the future, and its function in the plant is more powerful than the commonly used strong activation domain (such as VP16 and EDLL). HighlightTAC3 is the first transcriptional activation domain identified from Chinese fir and its function is more powerful than some commonly used strong transcriptional activators (such as VP16 and EDLL)

BackgroundHevea brasiliensis is the main global source of natural rubber. Due to fungal disease pressure in hot, humid regions, rubber plantations have been moved to drier "escape areas" with lower temperatures. In order to analyze gene expression regulation during cold exposure, we studied young GT1 and RRIM 600 rubber tree clones with different cold tolerance strategies. ResultsAlongside traditional differential expression approaches, an RNA-seq gene coexpression network (GCN) was developed with 27,220 genes grouped into 205 gene clusters. The GCN related most rubber tree cold stress molecular responses to 31 clusters across three GCN modules: a downregulated group with 16 clusters and two upregulated groups with twelve and three clusters. The hub genes of the cold-responsive modules were also identified and analyzed. We observed that the general response to short-term cold exposure involves complex regulation of the jasmonic acid (JA) stress response and programmed cell death (PCD), upregulation of ethylene-responsive genes, and relaxation of florigen gene inhibition. As a result, we identified single DEGs and gained insights into the mechanisms involved in the response to cold stress in young rubber trees. ConclusionsOur findings may represent the species genetic stress responses developed during the course of evolution, since the examined varieties were genotypes selected during the early years of rubber tree domestication. Understanding the cold response mechanisms in H. brasiliensis could improve breeding strategies for this crop, which has a narrow genetic base, is being impacted by climate change and is the only source for large-scale rubber production.

Changes in growth environment trigger stress responses in most organisms. The mechanisms mediating these responses are only partly understood and involve signaling pathways and transcription factors. Mediator is a conserved co-regulator complex required for transcriptional regulation of all eukaryotic protein-encoding genes. However, its function in abiotic stress responses is elusive. We here describe global gene expression changes triggered by salt stress in Arabidopsis. To explore the involvement of Mediator in salt stress response we characterized med9, med16, med18, and cdk8 mutants representing each of the four modules of Mediator. Our transcriptome data revealed enrichment of shared and specific cis-elements corresponding to unique transcription factors in promoters of mis-regulated genes for each mutant. We show that individual Mediator subunits interact with specific transcription factors to generate a transcriptional stress response and that the mutant phenotypes support the transcriptome data. med16 and med18, and to some extent cdk8, display defects in abscisic acid and anthocyanin metabolism and we identify signal molecules, transcription factors and target genes involved in these pathways as dysregulated in the Mediator mutants. Our results reveal how signals from different stress response pathways are dependent on and integrated by Mediator subunits to coordinate a functional response to salt stress.

Biotic and abiotic environmental factors shape plant responses. As such the interplay between viral infection and heat-stress can trigger specific physiological and metabolic plant responses that lead to gene-specific changes in defense and development. However, although plant gene expression patterns have been thoroughly studied under a single stress, the extent to which the combination of both stressors could modulate common or exclusive signaling pathways remains unclear. In this study, we examined the effects of watermelon mosaic virus infection and diurnal temperature variations (20/14 {degrees}C, 26/20 {degrees}C, and 32/24 {degrees}C) on the gene responses of two plant species (melon and zucchini), each with high- and low-temperature tolerance, using a differential 3mRNA-seq approach. The WMV load was much greater in zucchini than in melon plants, and was also dependent on the temperature conditions and tolerance of each plant species. Our comparative RNA-seq analysis revealed that the percentage of differentially expressed genes (DEGs) was higher in the thermo-susceptible plants of both species under the combination of WMV infection and low temperatures (20 {degrees}C). Among these significantly regulated genes, between 37 % and 45 % were related to biotic and/or abiotic stress. Furthermore, we found that 30 GO terms were involved in the response to both combined stress from low temperatures and 23 GO terms for high temperatures, which were exclusive to the thermotolerant varieties. Together, these findings allowed the identification of two unique orthologous genes linked to temperature and virus infection in melon and zucchini plants. Understanding the effects of biotic and abiotic factors on plant responses is essential for unraveling the complexity of plant-pathogen-environment interactions and developing strategies to enhance plant resilience and productivity under changing climatic conditions.

In plants, 14-3-3 proteins are recognized as mediators of signal transduction and function in both development and stress response. However, their functions have not been reported in the C4 crop foxtail millet. Here, phylogenetic analysis categorized foxtail millet 14-3-3s (SiGRFs) into ten discrete groups (Clusters I to {square}). Transcriptome and qPCR analyses showed that all the SiGRFs responded to at least one abiotic stress. All but one SiGRF-overexpressing (OE) Arabidopsis thaliana line (SiGRF1) exhibited insensitivity to abiotic stresses during seed germination and seedling growth. Compared with the Col-0 wild-type, SiGRF1-OEs had slightly lower germination rates and smaller leaves. However, flowering time of SiGRF1-OEs occurred earlier than that of Col-0 under high-salt stress. Interaction of SiGRF1 with a foxtail millet E3 ubiquitin-protein ligase (SiRNF1/2) indicates that the proteinase system might hydrolyse SiGRF1. Further investigation showed that SiGRF1 localized in the cytoplasm, and its gene was ubiquitously expressed in various tissues throughout various developmental stages. Additionally, flowering-related genes, WRKY71, FLOWERING LOCUS T, LEAFY and FRUITFULL, in SiGRF1-OEs exhibited considerably higher expression levels than those in Col-0 under salinity-stressed conditions. Results suggest that SiGRF1 hastens flowering, thereby providing a means for foxtail millet to complete its life cycle and avoid further salt stress. HighlightSiGRFs in foxtail millet: SiGRF1 hastens flowering in transgenic Arabidopsis thaliana exposed to salt stress

Despite being widespread in plants, endogenous pararetroviruses (EPRVs) are still poorly understood in barley and many other cereal crops. In this study, the barley reference genome was examined and from that a new EPRV was identified and named Hvu-EPRV. In contrast to all EPRVs identified thus far, Hvu-EPRV contains long terminal repeats (LTRs) which are similar to LTR retrotransposons. Homologous sequences of Hvu-EPRV were found in a wide range of plants, however, only those in 17 grasses belonging to the six tribes contain LTRs. The insertion times of nested LTR retrotransposons indicated that Hvu-EPRVs inserted into barley more than 2.37 million years ago, but the invasion and endogenization of Hvu-EPRV related elements in the grass family may be ancient, and horizontal transfers may have occurred between grasses. Phylogenetic analysis revealed that Hvu-EPRV and its homologs in grasses were grouped apart from all 13 reported genera of exogenous and endogenous pararetroviruses, thus the EPRVs in grasses represent a novel genus of the Caulimoviridae family named Moridahovirus. Genome-wide comparisons of Hvu-EPRVs were conducted between the reference genome and other 84 genomes of cultivated and wild barley, three independent integration events were observed and suggested that the integrations likely occurred after the divergence between barley and its wild progenitor. This is the first time to identify EPRVs with LTRs and to detect their recent integrations, and this research provides new insights into the evolution of plant EPRVs and their invasion history in the grass family.

Vigna luteola, a wild legume species, shows remarkable variation in salinity tolerance across its natural habitats, with coastal populations exhibiting high tolerance and riverbank populations being sensitive. This intraspecific variation provides a valuable system for investigating the genetic basis of salt tolerance. A major QTL for salt tolerance was previously identified by crossing salt-tolerant and salt-sensitive accessions, but the responsible genes remain unknown. In this study, grafting experiments between the two accessions revealed that the root plays a primary role in salt tolerance by suppressing Na transport to the shoot. We then conducted root transcriptome analysis and identified four candidate genes located within the QTL and highly expressed under salt stress in the tolerant accession: CBL-INTERACTING PROTEIN KINASE 6 (CIPK6), CAFFEOYL SHIKIMATE ESTERASE (CSE), FCS-LIKE ZINC FINGER PROTEIN 13 (FLZ13), and DROUGHT-INDUCED 21 (DI21). Promoter analysis revealed that the CIPK6 promoter contains transcription factor binding motifs unique to the salt-tolerant accession, which may contribute to its high expression under salt stress. These findings suggest that CIPK6 is regulated by cis-regulatory differences and is the most promising candidate for the salt-tolerance QTL. The identified genes in this study provide a foundation for developing salt-tolerant crops in the future.

Perturbations in ribosome biogenesis cause a type of cellular stress called nucleolar or ribosomal stress, which triggers adaptive responses in both animal and plant cells. The Arabidopsis ANAC082 transcription factor has been identified as a key mediator of the plant nucleolar stress response. The 5'-untranslated region (5'-UTR) of ANAC082 mRNA contains an upstream ORF (uORF) encoding an evolutionarily conserved amino acid sequence. Here, we report that this uORF mediates the upregulation of ANAC082 translation in response to nucleolar stress. When transgenic Arabidopsis plants containing a luciferase reporter gene under the control of the ANAC082 promoter and 5'-UTR were treated with reagents that induced nucleolar stress, translation of the reporter gene was enhanced in a uORF sequence-dependent manner. Additionally, we examined the effect of an endoplasmic reticulum (ER) stress-inducing reagent on reporter gene expression because the closest homolog of ANAC082 in Arabidopsis, ANAC103, is involved in the ER stress response. However, the ANAC082 uORF did not respond to ER stress. Interestingly, although ANAC103 has a uORF with an amino acid sequence similar to that of the ANAC082 uORF, the C-terminal sequence critical for regulation is not well conserved among ANAC103 homologs in Brassicaceae. Transient expression assays revealed that unlike the ANAC082 uORF, the ANAC103 uORF does not exert a sequence-dependent regulatory effect. Altogether, our findings suggest that the ANAC082 uORF is important for the nucleolar stress response but not for the ER stress response, and that for this reason, the uORF sequence-dependent translational regulation was lost in ANAC103 during evolution.

Beet curly top Iran Virus (BCTIV) is a yield-limiting geminivirus belonging to the becurtovirus genus. The genome organization of BCTIV is unique such that the complementary strand of BCTIV resembles mastreviruses, whereas the virion strand organization is close to curtoviruses. Geminiviruses are known to avoid the plant defense system by suppressing the RNA interference mechanisms both at the transcriptional gene silencing (TGS) and post-transcriptional gene silencing (PTGS) level. Multiple geminivirus genes have been identified as viral suppressors of RNA silencing (VSR) but VSR activity remains elusive in becurtoviruses. By screening all verified open reading frames in the BCTIV genome, we found that only V2 and Rep were able to suppress specific PTGS mechanisms, triggered by the expression of a partial or full-length sense-strand transcript of the target gene (S-PTGS). BCTIV-V2 could suppress S-PTGS more efficiently than BCTIV-Rep when then the target GFP gene is transiently expressed. On the other hand, S-PTGS is suppressed by Rep but not V2 when target GFP is only stably expressed. Deletional mutagenesis of BCTIV-Rep implicated that multiple domains are required for its VSR activity. Furthermore, neither V2 nor Rep could fully suppress local PTGS induced by inverted repeat targeting GFP (GFP-IR). Also, in a closer look at the spread of local silencing by GFP-IR, we observed that V2 or Rep are not able to suppress the movement of sRNAs. Nevertheless, Rep suppressed the systemic silencing induced by GFP-IR in 16C plants. Northern blot analyses showed that BCTIV-Rep inhibits silencing by mitigating sRNA production, whereas BCTIV-V2 does not alter sRNA levels. In summary, both the silencing phenotype and the molecular signatures of silencing implicate distinct modes of VSR activity of BCTIV-Rep and -V2.

Long non-coding RNAs (lncRNAs) perform prominent role in the regulation of gene expression during plant development and stress response by directly interacting with DNA, RNA, proteins, and/or triggering production of small regulatory RNA molecules. The objective of our study is to understand the systems-level response of the same plant species to highly diverse pathogens across different kingdoms and evaluate the patterns of similarity vs differences, specifically in the context of lncRNAs. Towards this objective, we performed a comparative in silico analysis of lncRNAs of Rice that are differentially expressed in response to infection by bacteria (Xanthomonas oryzae), fungus (Magnaporthe oryzae) and virus (Rice black dwarf virus). Using a tailored lncRNA analysis pipeline, we successfully identified 1125, 719 and 240 lncRNAs in Xanthomonas oryzae infection susceptible cultivar CT9737-6-1-3P-M, Magnaporthe oryzae susceptible LTH accession, and Rice black streaked dwarf virus susceptible Wuyujing No. 7 rice cultivars respectively. The in-silico predicted Cis- and Trans-target genes of lncRNAs were subsequently used to identify the pathways modulated by these lncRNA and how they cluster into unique categories of plant responses to pathogen infections. To further substantiate the role of predicted lncRNAs in plant defence and immune response our analysis finds that many of the lncRNAs co-localize with the QTLs associated with Blast and Bacterial blight resistance in rice. Our in silico analysis provides a list of common and unique pathogen specific lncRNAs that can provide vital insights into the generic vs tailored mechanisms adopted by rice in different infection scenarios.

APETALA2 (AP2)/ERF family transcription factors (TFs) contribute an important function against various external cues. Our study reports AtERF60, an AP2/ERF TF, which plays a key role in regulating the ABR1 gene, leading to an altered basal resistance response in Arabidopsis. AtERF60 is induced in response to drought, salt, abscisic acid (ABA), salicylic acid (SA), and bacterial pathogen PstDC3000 infection. AtERF60 interacts with DEHYDRATION RESPONSE ELEMENTS and GCC box, indicating its ability to regulate multiple responses. The overexpressing lines of AtERF60 demonstrated increased resistance to PstDC3000 infection, whereas erf60 mutant lines showed increased susceptibility. Complementation of the erf60 mutant background exhibits no significant difference towards PstDC3000 infection compared with the WT Col-0. Microarray and qRT-PCR analysis of overexpression and mutant lines indicated that AtERF60 regulates stress-inducible genes. The induction of these differentially expressed transcripts was significantly increased in erf60 mutant lines, whereas it was reversed in wild-type lines when AtERF60 was complemented. ABR1 was one of the differentially expressed transcripts, and we discovered that AtERF60 interacts with the DRE cis-elements in the ABR1 promoter. Further, qRT-PCR expression analysis after infection with PstDC3000 in AtERF60-OX, erf60 and its complementation background suggest that the mutation in AtERF60 upregulates ABR1 activity, leading to the enhanced susceptibility towards PstDC3000. Conversely, AtERF60 overexpression suppresses ABR1 activity, strengthening the basal resistance responses of Arabidopsis.

Epigenetic regulation has emerged as a significant element in adaptation to heterogeneous and stressful environments, with modifications in DNA methylation being particularly relevant in plants. DNA methylation inhibitors have been used to investigate the relationship between DNA methylation and plastic plant phenotypes. However, their effect in gene expression regulation along lifetime remains understudied in non-model plants. Here, we analyze the effects of seed exposure to 5-azacytidine (5-azaC) in plant gene regulation. Scarified seeds from a single inbred line of Erodium cicutarium were soaked for 48 h in either water or a low concentration solution of 5-azaC before sowing. Subsequently, RNA was extracted from juvenile roots, juvenile leaves and adult leaves, and their transcriptomes were sequenced. Differential gene expression analysis was performed between treatments (control vs. treated) for all tissues together and separately. Beforehand, a draft genome of E. cicutarium was assembled to use it as reference for the transcriptome analysis, and its DNA methyltransferase genes were characterized. We found that 5-azaC up-regulated chromomethylase CMT1 across all treated samples, and the domain rearranged DNA methyltransferase DRM2 in juvenile roots. Furthermore, adult leaves showed more differentially expressed genes between control and 5-azaC treated samples compared to juvenile leaves, supporting long term transcriptomic effects of a short exposure to 5-azaC at seed germination. At adult stage, leaves of individuals treated with 5-azaC exhibited up-regulation of genes involved in seed dormancy release, photoinhibition, and osmotic stress. Finally, gene co-expression network analysis revealed a module of co-expressed genes with differential gene expression linked to 5-azaC treatment in juvenile roots, that was enriched with genes involved in retrotransposon activity and in anthocyanin metabolism. Altogether, this study illustrates how the experimental treatment with 5-azaC at seed stage generates tissue- and age-specific transcriptional shifts, directly affecting gene regulation and potentially broadening phenotype variation in this fast-growing annual plant.

Wheat yellow mosaic virus (WYMV) causes severe viral wheat disease in Asia. The WYMV P1 protein encoded by RNA2 has viral suppressor of RNA silencing (VSR) activity to facilitate virus infection; however, VSR activity has not been identified for P2 protein encoded by RNA2. In this study, P2 protein exhibited strong VSR activity in Nicotiana benthamiana at the four-leaf stage, and point mutants P70A and G230A lost VSR activity. Protein P2 interacted with calmodulin (CaM) protein, a gene-silencing associated protein, while point mutants P70A and G230A did not interact with it. Competitive bimolecular fluorescence complementation and competitive co-immunoprecipitation experiments showed that P2 interfered with the interaction between CaM and calmodulin-binding transcription activator 3 (CAMTA3), but the point mutants P70A and G230A could not. Mechanical inoculation of wheat with in vitro transcripts of WYMV infectious cDNA clone further confirmed that VSR-deficient mutants P70A and G230A decreased WYMV infection in wheat plants compared with the wild type. In addition, RNA silencing, temperature, and autophagy had significant effects on accumulation of P2 protein in N. benthamiana leaves. In conclusion, WYMV P2 plays a VSR role in wheat and promotes virus infection by interfering with calmodulin-related antiviral RNAi defense. One-sentence summaryWYMV P2 protein exerts VSR activity by interfering with the CaM-CAMTA3 interaction to facilitate virus efficient systemic infection in wheat plants.

BackgroundA significant family of transcription factors known as WRKY genes include many physiological functions and environmental adaptations. However, insufficient information was previously available about the WRKY genes in Lagenaria siceraria, a crucial crop with substantial economic significance. The recent publication of the whole-genome sequence of L. siceraria has allowed us to perform a genome-wide investigation of the organization of the WRKY genes in L. siceraria. ResultsIn the present study, 57 L. siceraria WRKY (LsiWRKY) genes were identified and given new names based on their relative chromosomal distribution. The 57 LsiWRKYs were further divided into three major groups and several subgroups based on their structural and phylogenetic properties. Segmentation duplication events have played a major role in the expansion of the WRKY gene family in L. siceraria. Phylogenetic comparisons of the Group III WRKY genes provide valuable insights into the evolutionary characteristics of WRKY genes in L. siceraria. Additionally, RNA-seq analysis revealed distinct expression pattern of WRKY genes across different tissues. ConclusionsThis study presents a preliminary analysis of the WRKY gene family in L. siceraria, including their structural characteristics, evolutionary traits, and tissue-specific expression patterns. The systematic insights provided here serve as a foundation for further functional studies aimed at enhancing L. siceraria crops. This knowledge holds promise for improving the cultivation and yield of L. siceraria, thereby contributing to agricultural advancements.

One of the most promising strategies of Pine Pitch Canker (PPC) management is the use of reproductive plant material resistant to the disease. Understanding the complexity of plant transcriptome that underlies the defence to the causal agent Fusarium circinatum, would greatly facilitate the development of an accurate breeding program. Long non-coding RNAs (lncRNAs) are emerging as important transcriptional regulators under biotic stresses in plants. However, to date, characterization of lncRNAs in conifer trees has not been reported. In this study, transcriptomic identification of lncRNAs was carried out using strand-specific paired-end RNA sequencing, from Pinus radiata samples inoculated with F. circinatum at an early stage of infection. Overall, 13,312 lncRNAs were predicted through a bioinformatics approach, including long intergenic non-coding RNAs (92.3%), antisense lncRNAs (3.3%) and intronic lncRNAs (2.9%). Compared with protein-coding RNAs, pine lncRNAs are shorter, have lower expression, lower GC content and harbour fewer and shorter exons. A total of 164 differentially expressed (DE) lncRNAs were identified in response to F. circinatum infection in the inoculated versus mock-inoculated P. radiata seedlings. The predicted cis-regulated target genes of these pathogen-responsive lncRNAs were related to defence mechanisms such as kinase activity, phytohormone regulation, and cell wall reinforcement. Co-expression network analysis of DE lncRNAs, DE protein-coding RNAs and lncRNA target genes also indicated a potential network regulating pectinesterase activity and cell wall remodelling. This study presents the first analysis of conifer lncRNAs involved in the regulation of defence network and provides the basis for future functional characterizations of lncRNAs in relation to pine defence responses against F. circinatum.

MiRNAs regulate plants responses to fungal infection and immunity by modulating the gene expression. Despite extensive works on miRNAs role during plant-fungus interaction, work in Capsicum annuum-Phytophthora capsici pathosystem is limited. Therefore, in the current study, genome-wide known and novel miRNAs were identified in two contrasting chilli pepper landraces, i.e. GojamMecha_9086 (resistant) and Dabat_80045 (susceptible) during P. capsici infection. The small RNA deep sequencing resulted in 79 known miRNAs corresponding to 24 miRNAs families and 477 novel miRNAs along with 22,895 potential targets, including 30 defence-related genes against P. capsici infection. The expression analysis of [~]29 known & 157 novel miRNAs in resistant and 30 known and 176 novel miRNAs in susceptible landrace revealed differential accumulation pattern. RT-qPCR of a set of 8 defence related miRNAs representing 4 novel (Pz-novel-miR428-1, Pz-novel-miR160-1, Pz-novel-miR1028-1, Pz-novel-miR204-1) and 4 known (Pz-known-miR803-1, Pz-known-miR2059-1, Pz-known-miR2560-1, Pz-known-miR1872-1) revealed differential accumulation pattern in both resistant and susceptible landrace. Additionally, validation of 8 target genes of corresponding miRNAs using RA-PCR, which as good as 5 RLM-RACE, revealed an inverse relation with their corresponding miRNAs suggesting their key role during disease response. This study provides comprehensive genome-wide information about the repertoire of miRNAs and their target genes expressed in resistant and susceptible chilli pepper landrace, which can serve as a valuable resource for better understanding the post-transcriptional regulatory mechanism during C. annuum - P. capsici pathosystem.

Heat shock factors (HSFs) are at the core of heat stress (HS) response in plants. However, the contribution of HSFs governing the inherent thermotolerance mechanism in tomato from sub-tropical hot climates is poorly understood. With the above aim, comparative expression profiles of the HSF family in a HS tolerant (CLN1621L) and a sensitive cultivar (CA4) of tomato under HS revealed cultivar-biased regulation of an activator (HSFA7a) and repressor (HSFB4a) class HSF. Functional characterization of HSFA7a that was strongly up-regulated in the tolerant cultivar by VIGS-based silencing and overexpression established it as a positive regulator of HS-tolerance. While knock-down and overexpression analyses of HSFB4a that was down-regulated in CLN1621L in HS, showed it as a negative regulator of thermotolerance. Promoter:GUS reporter assays and promoter sequence analyses suggest heat-mediated transcriptional control of both the HSF genes in the contrasting cultivars. Moreover, we show HSFB4a is also regulated post-transcriptionally by microRNA Sly-miR4200 using degradome, short-tandem-target-mimic of Sly-miR4200 and transient in-planta Sly-miR4200-effector:HSFB4a-reporter assays. This miRNA is induced several folds upon HS in the tolerant variety thereby reducing HSFB4a levels. We thus propose that the alleviation of HSFB4a repressor governs thermotolerance in the tolerant cultivar by regulating downstream heat stress responsive genes.

Geminiviruses are a large group of plant viruses responsible for yield loss in various crops, mainly in the tropical and sub-tropical regions. Geminiviruses encode six to nine multifunctional proteins, which interact with plant components to cause pathogenesis. One of the least studied geminiviral proteins is AC5. This study presents the first evidence of an AC5 protein interacting with a component of the abscisic acid signalling pathway, resulting in a depressed state. We show that the AC5 protein, encoded by Sri Lankan cassava mosaic virus is important for symptom development and virus accumulation in the experimental host Nicotiana benthamiana. The above interaction perturbs the abscisic acid signalling pathways, leading to compromised expression of defense-related genes and insensitivity to abscisic acid in transgenic Arabidopsis plants. This suggests a novel role of AC5 to facilitate virus propagation. Furthermore, we show that transiently suppressing the expression in N. benthamiana PP2C with which AC5 interacts, results in a reduction in viral titers possibly due to augmented ABA signaling and its defense-related roles. This research provides valuable insights into how geminiviral proteins manipulate ABA-mediated defence pathways, paving the way for further investigation into the underlying mechanisms and potential applications in plant protection against viral infections.

Multistep phosphorelay (MSP) signaling integrates hormonal and environmental signals to control plant development and adaptive responses. The type-A RESPONSE REGULATORs (RRAs), the downstream members of the MSP cascade and cytokinin primary response genes, are supposed to mediate primarily the negative feedback regulation of (cytokinin-induced) MSP signaling. However, the transcriptional data suggest the involvement of RRAs in stress-related responses as well. By employing evolutionary conservation with the well-characterized Arabidopsis thaliana RRAs, we identified 5 and 38 novel putative RRAs in Brassica oleracea and Brassica napus, respectively. Our phylogenetic analysis suggests the existence of gene-specific selective pressure, maintaining the homologs of ARR3, ARR6, and ARR16 as singletons during the evolution of Brassica oleracea and Brassica rapa. We categorized RRAs based on the kinetics of their cytokinin-mediated upregulation and observed both similarities and specificities in this type of response across Brassicaceae. Using bioinformatic analysis and experimental data demonstrating the cytokinin responsiveness of Arabidopsis-derived TCSv2 reporter, we unveil the mechanistic conservation of cytokinin-mediated upregulation of RRAs in Brassica rapa and Brassica napus. Notably, we identify partial cytokinin dependency of cold stress-induced RRA transcription, thus corroborating the role of cytokinin signaling in the crop adaptive responses. HighlightsWe identified Brassica homologs of Arabidopsis type-A response regulators (RRAs), demonstrate existence of selective pressure preventing several RRAs multiplication during Brassicaceae evolution and describe cytokinin dependency of cold-induced RRAs upregulation.

The intricate molecular interplay between beneficial fungi and plants is vital to plant growth promotion and induced defense response. This study explored the role of DCL-mediated RNA silencing in the interaction between the biocontrol fungus Clonostachys rosea and wheat roots. We investigated the impact of DCL (Dicer-like) gene deletions in C. rosea on its root colonization ability. Our results revealed that the deletion of dcl2 significantly enhanced C. rosea biomass on wheat roots, indicating a pivotal role of DCL2 in root colonization. Transcriptome sequencing of C. rosea and wheat during their interactions unveiled extensive gene expression changes. In wheat, genes related to stress responses were upregulated during C. rosea interactions, while genes associated with plant cell wall modification and metabolic processes were downregulated, suggesting complex regulatory responses and a trade-off between defense mechanisms and growth promotion. Deletion of C. rosea dcl1 and dcl2 altered the transcriptomic responses of wheat roots during interactions. Wheat genes associated with stress responses were downregulated during interactions with DCL deletion strains. In contrast, genes involved in metabolic processes and growth were upregulated, emphasizing the cross-kingdom regulatory role of C. rosea small RNAs (sRNAs). We identified 18 wheat miRNAs responsive to C. rosea interactions. Furthermore, we identified 24 endogenous and six cross-kingdom potential gene targets for seven and five differentially expressed miRNAs, supported by their inverse gene expression pattern. In C. rosea, we found a large transcriptional reprogramming of genes during interaction with wheat roots. The upregulated genes were associated with carbohydrate and polysaccharide catabolic processes, membrane transporters and effectors. Conversely, downregulated genes were mainly associated with transition metal ion transport and homeostasis processes. The deletion of dcl1 and dcl2 had significant effects on gene expression. A higher number of genes upregulated in WT during the interaction were restored in DCL deletion mutants, suggesting DCL-mediated gene expression regulation. Furthermore, we identified 21 differentially expressed micro-RNA-like RNAs (milRNAs) in C. rosea; nine were DCL-dependent. They had putative gene targets in C. rosea, including transcription factors, effectors, transporters, and enzymes involved in specialized metabolite production. Cross-kingdom RNA silencing was also observed, with seven DCL-dependent C. rosea milRNAs potentially targeting 29 genes in wheat. These findings provide valuable insights into the molecular mechanisms underlying the beneficial interaction between fungi and plant roots. In addition, the study shed light on the role of sRNA-mediated gene regulation in the C. rosea-wheat interaction, with potential implications for sustainable agriculture and biocontrol strategies.