Xiuhua Xue

Mitogen-activated protein kinases 3 and 6 (MPK3/MPK6) are central to pattern-triggered immunity (PTI) and effector-triggered immunity (ETI) in Arabidopsis, yet the involvement of long noncoding RNAs (lncRNAs, >200 nt) in these pathways is poorly understood. Here, transcriptomic analyses were performed to compare lncRNA and protein-coding gene (PCG) expression profiles in wild-type (WT) and MPK3/MPK6-deficient (MPK6SR) Arabidopsis plants. These plants were inoculated with either Pseudomonas syringae pv. tomato (Pst) DC3000, which elicits both PTI and ETI, or its type III secretion-deficient mutant, Pst DC3000 hrcC⁻, which induces only PTI. RNA sequencing (RNA-seq) analysis of 18 samples identified 1388 known and 70 novel lncRNAs, among which differentially expressed lncRNAs (DElncRNAs) involved in disease resistance were further identified. Using integrative analyses, including weighted gene co-expression network analysis (WGCNA), prediction of lncRNA cis-regulatory targets for PCGs, and validation via reverse transcription-quantitative PCR (RT-qPCR), three core lncRNA-mediated regulatory modules were identified: (i) MPK3/MPK6-dependent PTI and ETI, where lncRNAs amplify signals; (ii) MPK3/MPK6-dependent PTI, where lncRNAs fine-tune basal immunity; and (iii) MPK3/MPK6-independent PTI and ETI, where lncRNAs serve as a backup regulatory network. These modules form a multi-layered immune regulatory network via cis- and trans-regulation and further enable the identification of lncRNA-PCG pairs involved in both regulatory modes. This work enhances the understanding of the molecular mechanisms underlying plant innate immunity.

Long non-coding RNAs (lncRNAs) are involved in plant biotic and abiotic stress responses, in which Ca²⁺ also plays a significant role. There is diversity in the regulation of different gene expressions by cytosolic Ca²⁺ ([Ca²⁺]_cyt) and nucleosolic Ca²⁺ ([Ca²⁺]_nuc). However, no studies have yet explored the interrelationship between lncRNAs and calcium signaling, nor how calcium signaling regulates the expression of lncRNAs. Here, we use transgenic materials PV-NES and NLS-PV, which simulate [Ca²⁺]_cyt- and [Ca²⁺]_nuc-deficient mutants, respectively, and wild type (WT) materials in response to hyperosmolarity (250 mM sorbitol) or salt stresses (125 mM NaCl) at different time points to obtain RNA-seq data, respectively. Then, we proceed with the screening of lncRNAs, adding 688 new lncRNAs to the known Arabidopsis lncRNA database. Subsequently, through the analysis of differentially expressed lncRNA genes, it was found that cytosolic or nucleosolic calcium signals have distinct regulatory effects on differentially expressed lncRNAs (DElncRNAs) and differentially expressed protein-coding genes (DEPCGs) treated with high-concentration NaCl and sorbitol at different times. Furthermore, through weighted correlation network analysis (WGCNA), it is discovered that under hyperosmolarity and salt stresses, lncRNA-associated PCGs are related to the cell wall structure, the plasma membrane component, and osmotic substances through trans-regulation. In addition, by screening for cis-regulatory target PCGs of Ca²⁺-regulated lncRNAs related to osmotic stress, we obtain a series of lncRNA-PCG pairs related to water transport, cell wall components, and lateral root formation. Therefore, we expand the existing Arabidopsis lncRNA database and obtain a series of lncRNAs and PCGs regulated by [Ca²⁺]_cyt or [Ca²⁺]_nuc in response to salt and hyperosmolarity stress, providing a new perspective for subsequent research on lncRNAs. We also explore the trans- and cis-regulated target PCGs of lncRNAs regulated by calcium signaling, providing new insights for further studying salt stress and osmotic stress.

Intrinsically disordered proteins (IDPs) include two types of proteins: partial disordered regions (IDRs) and wholly disordered proteins (WDPs). Extensive studies focused on the proteins with IDRs, but less is known about WDPs because of their difficult-to-form folded tertiary structure. In this study, we developed a bioinformatics method for screening more than 50 amino acids in the genome level and found a total of 27 categories, including 56 WDPs, in Arabidopsis. After comparing with 56 randomly selected structural proteins, we found that WDPs possessed a more wide range of theoretical isoelectric point (PI), a more negative of Grand Average of Hydropathicity (GRAVY), a higher value of Instability Index (II), and lower values of Aliphatic Index (AI). In addition, by calculating the FCR (fraction of charged residue) and NCPR (net charge per residue) values of each WDP, we found 20 WDPs in R1 (FCR < 0.25 and NCPR < 0.25) group, 15 in R2 (0.25 ≤ FCR ≤ 0.35 and NCPR ≤ 0.35), 19 in R3 (FCR > 0.35 and NCPR ≤ 0.35), and two in R4 (FCR > 0.35 and NCPR > 0.35). Moreover, the gene expression and protein-protein interaction (PPI) network analysis showed that WDPs perform different biological functions. We also showed that two WDPs, SIS (Salt Induced Serine rich) and RAB18 (a dehydrin family protein), undergo the in vitro liquid-liquid phase separation (LLPS). Therefore, our results provide insight into understanding the biochemical characters and biological functions of WDPs in plants.

Intrinsically disordered proteins (IDPs) include two types of proteins: one contains partial disordered regions (IDRs), the other is wholly disordered proteins (WDPs). Extensive studies focused on the proteins with IDRs, but less is known about WDPs because of their difficult to form the folded tertiary structure. In this study, we developed a bioinformatics method for screening WDPs more than 50 amino acids in the genome level and found a total of 27 categories including 56 WDPs in Arabidopsis. After comparing with randomly selected 56 structural proteins, we found that WDPs possessed more wide range of theoretical isoelectric point (PI), more negative of Grand Average of Hydropathicity (GRAVY), higher value of Instability Index (II) and lower values of Aliphatic Index (AI). In addition, by calculating the FCR (fraction of charged residue) and NCPR (net charge per residue) values of each WDP, we found twenty WDPs in R1 (FCR < 0.25 & NCPR < 0.25) group, fifteen in R2 (0.25 ≤ FCR ≤ 0.35 & NCPR ≤ 0.35), nineteen in R3 (FCR > 0.35 & NCPR ≤ 0.35), and two in R4 (FCR > 0.35 & NCPR > 0.35). Moreover, the gene expression and protein-protein interaction (PPI) network analysis showed that WDPs perform different biological functions. We also showed that two WDPs, SIS (Salt Induced Serine rich) and RAB18 (a dehydrin family protein) undergo the in vitro liquid-liquid phase separation (LLPS). Therefore, our results provide insight into understanding the biochemical characters and biological functions of WDPs in plants.

Due to their immobility and possession of underground parts, plants have evolved various mechanisms to endure and adapt to abiotic stresses such as extreme temperatures, drought, and salinity. However, the contribution of long noncoding RNAs (lncRNAs) to different abiotic stresses and distinct rice seedling parts remains largely uncharacterized beyond the protein-coding gene (PCG) layer. Using transcriptomics and bioinformatics methods, we systematically identified lncRNAs and characterized their expression patterns in the roots and shoots of wild type (WT) and ososca1.1 (reduced hyperosmolality-induced [Ca²⁺]_i increase in rice) seedlings under hyperosmolarity and salt stresses. Here, 2937 candidate lncRNAs were identified in rice seedlings, with intergenic lncRNAs representing the largest category. Although the detectable sequence conservation of lncRNAs was low, we observed that lncRNAs had more orthologs within the Oryza. By comparing WT and ososca1.1, the transcription level of OsOSCA1.1-related lncRNAs in roots was greatly enhanced in the face of hyperosmolality stress. Regarding regulation mode, the co-expression network revealed connections between trans-regulated lncRNAs and their target PCGs related to OsOSCA1.1 and its mediation of hyperosmolality stress sensing. Interestingly, compared to PCGs, the expression of lncRNAs in roots was more sensitive to hyperosmolarity stress than to salt stress. Furthermore, OsOSCA1.1-related hyperosmolarity stress-responsive lncRNAs were enriched in roots, and their potential cis-regulated genes were associated with transcriptional regulation and signaling transduction. Not to be ignored, we identified a motif-conserved and hyperosmolarity stress-activated lncRNA gene (OSlncRNA), speculating on its origin and evolutionary history in Oryza. In summary, we provide a global perspective and a lncRNA resource to understand hyperosmolality stress sensing in rice roots, which helps to decode the complex molecular networks involved in plant sensing and adaptation to stressful environments.

Long noncoding RNAs (lncRNAs) are RNA molecules longer than 200 nt, which lack the ability to encode proteins and are involved in multifarious growth, development, and regulatory processes in plants and mammals. However, the environmental-regulated expression profiles of lncRNAs in Orinus that may associated with their adaptation on the Qinghai-Xizang (Tibet) Plateau (QTP) have never been characterized. Here, we utilized transcriptomic sequencing data of two Orinus species (O. thoroldii and O. kokonoricus) to identify 1624 lncRNAs, including 1119 intergenic lncRNAs, 200 antisense lncRNAs, five intronic lncRNAs, and 300 sense lncRNAs. In addition, the evolutionary relationships of Orinus lncRNAs showed limited sequence conservation among 39 species, which implied that Orinus-specific lncRNAs contribute to speciation adaptation evolution. Furthermore, considering the cis-regulation mechanism, from 286 differentially expressed lncRNAs (DElncRNAs) and their nearby protein coding genes (PCGs) between O. thoroldii and O. kokonoricus, 128 lncRNA-PCG pairs were obtained in O. thoroldii, whereas 92 lncRNA-PCG pairs were obtained in O. kokonoricus. In addition, a total of 19 lncRNA-PCG pairs in O. thoroldii and 14 lncRNA-PCG pairs in O. kokonoricus were found to participate in different biological processes, indicating that the different expression profiles of DElncRNAs between O. thoroldii and O. kokonoricus were associated with their adaptation at different elevations on the QTP. We also found several pairs of DElncRNA nearby transcription factors (TFs), indicating that these DElncRNAs regulate the expression of TFs to aid O. thoroldii in adapting to the environment. Therefore, this work systematically identified a series of lncRNAs in Orinus, laying the groundwork for further exploration into the biological function of Orinus in environmental adaptation.

Many plants have the capability to accumulate anthocyanins for coloration, and anthocyanins are advantageous to human health. In the case of hulless barley (Hordeum vulgare L. var. nudum), investigation into the mechanism of anthocyanin formation is limited to the level of protein-coding genes (PCGs). Here, we conducted a comprehensive bioinformatics analysis to identify a total of 9414 long noncoding RNAs (lncRNAs) in the seed coats of purple and white hulless barley along a developmental gradient. Transcriptome-wide profiles of lncRNAs documented several properties, including GC content fluctuation, uneven length, a diverse range of exon numbers, and a wide variety of transcript classifications. We found that certain lncRNAs in hulless barley possess detectable sequence conservation with Hordeum vulgare and other monocots. Furthermore, both differentially expressed lncRNAs (DElncRNAs) and PCGs (DEPCGs) were concentrated in the later seed development stages. On the one hand, DElncRNAs could potentially cis-regulate DEPCGs associated with multiple metabolic pathways, including flavonoid and anthocyanin biosynthesis in the late milk and soft dough stages. On the other hand, there was an opportunity for trans-regulated lncRNAs in the color-forming module to affect seed coat color by upregulating PCGs in the anthocyanin pathway. In addition, the interweaving of hulless barley lncRNAs and diverse TFs may function in seed coat coloration. Notably, we depicted a dynamic portrait of the anthocyanin synthesis pathway containing hulless barley lncRNAs. Therefore, this work provides valuable gene resources and more insights into the molecular mechanisms underlying anthocyanin accumulation in hulless barley from the perspective of lncRNAs, which facilitate the development of molecular design breeding in crops.

Verticillium wilt is a kind of soil-borne plant fungal disease caused by Verticillium dahliae (Vd). Vd 991 is a strong pathogen causing cotton Verticillium wilt. Previously, we isolated a compound from the secondary metabolites of Bacillus subtilis J15 (BS J15), which showed a significant control effect on cotton Verticillium wilt and was identified as C17 mycosubtilin. However, the specific fungistatic mechanism by which C17 mycosubtilin antagonizes Vd 991 is not clear. Here, we first showed that C17 mycosubtilin inhibits the growth of Vd 991 and affects germination of spores at the minimum inhibitory concentration (MIC). Morphological observation showed that C17 mycosubtilin treatment caused shrinking, sinking, and even damage to spores; the hyphae became twisted and rough, the surface was sunken, and the contents were unevenly distributed, resulting in thinning and damage to the cell membrane and cell wall and swelling of mitochondria of fungi. Flow cytometry analysis with ANNEXINV-FITC/PI staining showed that C17 mycosubtilin induces necrosis of Vd 991 cells in a time-dependent manner. Differential transcription analysis showed that C17 mycosubtilin at a semi-inhibitory concentration (IC50) treated Vd 991 for 2 and 6 h and inhibited fungal growth mainly by destroying synthesis of the fungal cell membrane and cell wall, inhibiting its DNA replication and transcriptional translation process, blocking its cell cycle, destroying fungal energy and substance metabolism, and disrupting the redox process of fungi. These results directly showed the mechanism by which C17 mycosubtilin antagonizes Vd 991, providing clues for the mechanism of action of lipopeptides and useful information for development of more effective antimicrobials.

Background Information: In many cellular processes including cell division, the synergistic dynamics of actin filaments and microtubules play vital roles. However, the regulatory mechanisms of these synergistic dynamics are not fully understood. Proteins such as formins are involved in actin filament–microtubule interactions and Arabidopsis thaliana formin 14 (AtFH14) may function as a crosslinker between actin filaments and microtubules in cell division, but the molecular mechanism underlying such crosslinking remains unclear.Results: Without microtubules, formin homology (FH) 1/FH2 of AtFH14 nucleated actin polymerisation from actin monomers and capped the barbed end of actin filaments. However, in the presence of microtubules, quantitative analysis showed that the binding affinity of AtFH14 FH1FH2 to microtubules was higher than that to actin filaments. Moreover, microtubule‐bound AtFH14 FH1FH2 neither nucleated actin polymerisation nor inhibited barbed end elongation. In contrast, tubulin did not affect AtFH14 FH1FH2 to nucleate actin polymerisation and inhibit barbed end elongation. Nevertheless, microtubule‐bound AtFH14 FH1FH2 bound actin filaments and the bound actin filaments slid and elongated along the microtubules or elongated away from the microtubules, which induced bundling or crosslinking of actin filaments and microtubules. Pharmacological analyses indicated that AtFH14 FH1FH2 promoted crosslinking of actin filaments and microtubules in vivo. Additionally, co‐sedimentation and fluorescent dye‐labelling experiments of AtFH14 FH2‐truncated proteins in vitro revealed the essential motifs of bundling actin filaments or microtubules, which were 63–92 aa and 42–62 aa in the AtFH14 FH2 N‐terminal, respectively, and 42–62 aa was the essential motif to crosslink actin filaments and microtubules.Conclusions and Significance: Our results aid in explaining how AtFH14 functions as a crosslinker between actin filaments and microtubules to regulate their dynamics via different manners during cell division. They also facilitate further understanding of the molecular mechanisms of the interactions between actin filaments and microtubules.