Unraveling plant-microbe symbioses using single-cell and spatial transcriptomics.

Plant-microbe symbioses require intense interaction and genetic coordination to successfully establish in specific cell types of the host and symbiont. Traditional RNA-seq methodologies lack the cellular resolution to fully capture these complexities, but single-cell and spatial transcriptomics (ST) are now allowing scientists to probe symbiotic interactions at an unprecedented level of detail. Here, we discuss the advantages that novel spatial and single-cell transcriptomic technologies provide in studying plant-microbe endosymbioses and highlight key recent studies. Finally, we consider the remaining limitations of applying these approaches to symbiosis research, which are mainly related to the simultaneous capture of both plant and microbial transcripts within the same cells.

Single-cell analysis identifies genes facilitating rhizobium infection in Lotus japonicus.

Legume-rhizobium signaling during establishment of symbiotic nitrogen fixation restricts rhizobium colonization to specific cells. A limited number of root hair cells allow infection threads to form, and only a fraction of the epidermal infection threads progress to cortical layers to establish functional nodules. Here we use single-cell analysis to define the epidermal and cortical cell populations that respond to and facilitate rhizobium infection. We then identify high-confidence nodulation gene candidates based on their specific expression in these populations, pinpointing genes stably associated with infection across genotypes and time points. We show that one of these, which we name SYMRKL1, encodes a protein with an ectodomain predicted to be nearly identical to that of SYMRK and is required for normal infection thread formation. Our work disentangles cellular processes and transcriptional modules that were previously confounded due to lack of cellular resolution, providing a more detailed understanding of symbiotic interactions.

Factors Associated With Treatment-Related Changes in Voice Volume in People With Multiple Sclerosis.

BACKGROUND: Vocal disorders are frequent in people with multiple sclerosis (MS). Cognitive impairment, fatigue, depression, and other clinical characteristics can be associated with treatment effectiveness in rehabilitation. Finding baseline characteristics that identify those who are responding to treatment can help the clinical decision-making process, which can then help improve the effectiveness of voice treatment. We developed a model to identify factors associated with treatment-related improvement on voice intensity in people with MS. METHODS: Data are from a randomized controlled trial of the effects of voice therapy. Forty-four people with MS were enrolled and randomized to receive Lee Silverman Voice Treatment LOUD, specifically addressing voice intensity, or conventional speech-therapy group. Voice intensity (dB) was measured during monologue before and after treatment and was used to differentiate those who responded (posttreatment voice intensity > 60 dB) from those who did not. Possible associated factors were cognitive impairment, fatigue, depression, disability, and disease duration. Associations were assessed by univariate logistic regression and univariate and multivariate linear regressions. RESULTS: Mean ± SD monologue voice intensity is improved in the whole sample (before rehabilitation: 51.8 ± 4.2 dB; and after rehabilitation 57.0 ± 6.5 dB; P < .001), and 11 people with MS (27.5%) responded to treatment. Specificity of treatment was associated with the return to normal voice intensity (OR, 14.28; 95% CI, 12.17-309.56) and we found a linear association between voice improvement and the specificity of treatment (6.65 [SE = 1.54] dB; P < .05). Moreover, the analysis revealed a nonlinear association between improvement and fatigue, suggesting increased benefits for people with MS with moderate fatigue. Other factors were not significantly associated with treatment effectiveness. CONCLUSIONS: Moderate fatigue and the specificity of the intervention seem to be key factors associated with clinically relevant improvement in voice intensity even in people with MS with a high level of disability and long disease duration.

Effects of voice rehabilitation in people with MS: A double-blinded long-term randomized controlled trial.

BACKGROUND: Hypophonia is a prevailing problem in people with multiple sclerosis (PwMS). However, evidence supporting the effectiveness of voice rehabilitation is lacking. OBJECTIVE: The aim of this study was to identify the most effective method to reduce hypophonia. METHODS: In this randomized controlled trial, 44 PwMS were randomized to intensive and high-effort voice treatment groups, the LSVT-LOUD®, and conventional treatment group. Subjects received 16 treatments (4 sessions/week) lasting 45 minutes. The primary outcome was voice intensity (dB) in monologue, vocalization, and sentences while voice handicap index (VHI) measured voice self-perception. Outcomes were assessed by a blinded observer at baseline, post-treatment, and 15-month follow-up (FU). RESULTS: Linear models revealed a significant post-intervention between-group mean difference in favor of LSVT-LOUD for monologue: +6.3 dB (95% CI: 2.5 to 10.1); vocalization: +7.4 dB (95% CI: 2.3 to 12.5); and sentences: +9.5 dB (95% CI: 4.7 to 14.3). However, 43.7% PwMS in the LSVT-LOUD and 10% in the conventional treatment group obtained a full recovery of voice intensity (>60 dB) post-treatment, Fisher's test = 13.3, p < 0.01. However, these improvements were not maintained at FU. Between-group differences at VHI were -10.8 (95% CI: -21.2 to -0.4) and -11.3 (95% CI: -24.3 to -1.7) in favor of LSVT-LOUD at post and FU. CONCLUSION: LSVT-LOUD can be a valid treatment to increase voice intensity in PwMS. However, results suggest the need for FU interventions targeting maintenance.

Gut Microbiome and Degradation Product Formation during Biodegradation of Expanded Polystyrene by Mealworm Larvae under Different Feeding Strategies.

Polystyrene (PS) is a plastic polymer extensively used for food packaging. PS is difficult to decompose and has low recycling rates, resulting in its accumulation in the environment, in the form of microplastic particles causing pollution and harming oceans and wildlife. Degradation of PS by mealworms (Tenebrio molitor) has been suggested as a possible biological strategy for plastic contamination; however, the biodegradation mechanism of PS by mealworms is poorly understood. It is hypothesized that the gut microbiome plays an important role in the degradation of PS by mealworms. This study carried out a comparative analysis of the gut microbiome of Tenebrio molitor larvae under different feeding strategies, and of the formation of degradation compounds (monomers, oligomers). A diet of bran:PS at 4:1 and 20:1 ratios was tested. The diet with the low ratio of bran:PS led to the presence of higher amounts of these compounds, compared to that with the high ratio. In addition, it was demonstrated that the addition of H2O significantly improved the biodegradation of PS monomer and oligomer residues, which could be identified only in the frass. The protein and nitrogen contents in insects' biomass and frass varied amongst treatments. The diets resulted in differences in the gut microbiota, and three potential bacterial strains were identified as candidates involved in the biodegradation of PS.

EFFECTOR OF TRANSCRIPTION factors are novel plant-specific regulators associated with genomic DNA methylation in Arabidopsis.

Plant-specific EFFECTORS OF TRANSCRIPTION (ET) are characterised by a variable number of highly conserved ET repeats, which are involved in zinc and DNA binding. In addition, ETs share a GIY-YIG domain, involved in DNA nicking activity. It was hypothesised that ETs might act as epigenetic regulators. Here, methylome, transcriptome and phenotypic analyses were performed to investigate the role of ET factors and their involvement in DNA methylation in Arabidopsis thaliana. Comparative DNA methylation and transcriptome analyses in flowers and seedlings of et mutants revealed ET-specific differentially expressed genes and mostly independently characteristic, ET-specific differentially methylated regions. Loss of ET function results in pleiotropic developmental defects. The accumulation of cyclobutane pyrimidine dimers after ultraviolet stress in et mutants suggests an ET function in DNA repair.

RWP-RK domain-containing transcription factors control cell differentiation during female gametophyte development in Arabidopsis.

The formation of gametes is a prerequisite for any sexually reproducing organism in order to complete its life cycle. In plants, female gametes are formed in a multicellular tissue, the female gametophyte or embryo sac. Although the events leading to the formation of the female gametophyte have been morphologically characterized, the molecular control of embryo sac development remains elusive. We used single and double mutants as well as cell-specific marker lines to characterize a novel class of gene regulators in Arabidopsis thaliana, the RWP-RK domain-containing (RKD) transcription factors. Morphological and histological analyses were conducted using confocal laser scanning and differential interference contrast microscopy. Gene expression and transcriptome analyses were performed using quantitative reverse transcription-PCR and RNA sequencing, respectively. Our results showed that RKD genes are expressed during distinct stages of embryo sac development. Morphological analysis of the mutants revealed severe distortions in gametophyte polarity and cell differentiation. Transcriptome analysis revealed changes in the expression of several gametophyte-specific gene families (RKD2 and RKD3) and ovule development-specific genes (RKD3), and identified pleiotropic effects on phytohormone pathways (RKD5). Our data provide novel insight into the regulatory control of female gametophyte development. RKDs are involved in the control of cell differentiation and are required for normal gametophytic development.

Wheat Subtilisin/Chymotrypsin Inhibitor (WSCI) as a scaffold for novel serine protease inhibitors with a given specificity.

WSCI (Wheat Subtilisin/Chymotrypsin Inhibitor) is a small protein belonging to the Potato inhibitor I family exhibiting a high content of essential amino acid. In addition to bacterial subtilisins and mammalian chymotrypsins, WSCI inhibits chymotrypsin-like activities isolated from digestive traits of a number of insect larvae. In vivo, as suggested for many plant proteinase inhibitors, WSCI seems to play a role of natural defence against attacks of pests and pathogens. The functional region of WSCI, containing the inhibitor reactive site (Met48-Glu49), corresponds to an extended flexible loop (Val42-Asp53) whose architecture is somehow stabilized by a number of secondary interactions established with a small ß-sheet located underneath. The aim of this study was to employ a WSCI molecule as a stable scaffold to obtain recombinant inhibitors with new acquired anti-proteinase activity or, alternatively, inactive WSCI variants. A gene sequence coding for the native WSCI, along with genes coding for muteins with different specficities, could be exploited to obtain transformed non-food use plants with improved insect resistance. On the other hand, the genetic transformation of cereal plants over-expressing inactive WSCI muteins could represent a possible strategy to improve the nutritional quality of cereal-based foods, without risk of interference with human or animal digestive enzymes. Here, we described the characterization of four muteins containing single/multiple amino acid substitutions at the WSCI reactive site and/or at its proximity. Modalities of interaction of these muteins with proteinases (subtilisin, trypsin and chymotrypsin) were investigated by time course hydrolysis and molecular simulations studies.

Enhanced cytotoxic activity of a bifunctional chimeric protein containing a type 1 ribosome-inactivating protein and a serine protease inhibitor.

Both ribosome-inactivating proteins (RIPs) and plant proteinase inhibitors, belong to protein families known to regulate cellular homeostasis and likely involved in plant defense. Nevertheless the interest in these protein classes is due to their potential use for the treatment of several important human diseases such as cancer. Thus, in the present study, type 1 ribosome-inactivating protein and wheat subtilisin/chymotrypsin inhibitor, were engineered into a chimeric protein with cytotoxic action selective for murine tumor cells, while lacking any appreciable toxicity on murine normal cells. This chimeric protein selectively sensitizes to apoptotic death cells derived from Simian-virus-40-transformed mouse fibroblasts (SVT2 cells). The cytotoxicity of this new recombinant product has been detected also on three different human malignant cells. Therefore action on tumor cells of this protein could represent a potentially very attractive novel tool for anticancer drug design.

WCI, a novel wheat chymotrypsin inhibitor: purification, primary structure, inhibitory properties and heterologous expression.

A novel chymotrypsin inhibitor, detected in the endosperm of Triticum aestivum, was purified and characterized with respect to the main physical-chemical properties. On the basis of its specificity, this inhibitor was named WCI (wheat chymotrypsin inhibitor). WCI is a monomeric neutral protein made up of 119 residues and molecular mass value of 12,933.40 Da. Automated sequence and mass spectrometry analyses, carried out on several samples of purified inhibitor, evidenced an intrinsic molecular heterogeneity due to the presence of the isoform [des-(Thr)WCI], accounting for about 40% of the total sample. In vitro, WCI acted as a strong inhibitor of bovine pancreatic chymotrypsin as well as of chymotryptic-like activities isolated from the midgut of two phytophagous insects, Helicoverpa armigera (Hüb.) and Tenebrio molitor L., respectively. No inhibitory activities were detected against bacterial subtilisins, bovine pancreatic trypsin, porcine pancreatic elastase or human leukocyte elastase. The primary structure of WCI was significantly similar (45.7-89.1%) to those of several proteins belonging to the cereal trypsin/α-amylase inhibitor super-family and showed the typical sequence motif of this crowed protein group. The cDNA of the inhibitor (wci-cDNA) was isolated from wheat immature caryopses and employed to obtain a recombinant product in E. coli. Experimental evidences indicated that the recombinant inhibitor was localized in the inclusion bodies from which it was recovered as soluble and partially active protein by applying an appropriate refolding procedure. WCI reactive site localization, as well as its inhibitory specificity, was investigated by molecular modeling approach.